Electrokinetically-altered fluids comprising charge-stabilized gas-containing nanostructures

ABSTRACT

Particular aspects provide compositions comprising an electrokinetically altered oxygenated aqueous fluid, wherein the oxygen in the fluid is present in an amount of at least 25 ppm. In certain aspects, the electrokinetically altered oxygenated aqueous fluid comprises electrokinetically modified or charged oxygen species present in an amount of at least 0.5 ppm. In certain aspects the electrokinetically altered oxygenated aqueous fluid comprises solvated electrons stabilized by molecular oxygen, and wherein the solvated electrons present in an amount of at least 0.01 ppm. In certain aspects, the fluid facilitates oxidation of pyrogallol to purpurogallin in the presence of horseradish peroxidase enzyme (HRP) in an amount above that afforded by a control pressure pot generated or fine-bubble generated aqueous fluid having an equivalent dissolved oxygen level, and wherein there is no hydrogen peroxide, or less than 0.1 ppm of hydrogen peroxide present in the electrokinetic oxygen-enriched aqueous fluid.

CROSS REFERENCE TO RELATED APPLICATION(S)

This application is a continuation of U.S. patent application Ser. No.11/924,595 filed Oct. 25, 2007 (now issued as U.S. Pat. No. 7,919,534),which claims the benefit of priority to U.S. Provisional ApplicationNos. 60/862,904, filed Oct. 25, 2006, 60/862,955, filed Oct. 25, 2006,and 60/982,38, filed Oct. 24, 2007.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention is directed generally to mixing devices and moreparticularly to mixing devices that mix two or more materials betweensurfaces, including such as between a rotating rotor and a stationarystator.

2. Description of the Related Art

FIG. 1 provides a partial block diagram, partial cross-sectional view ofa prior art device 10 for diffusing or emulsifying one or two gaseous orliquid materials (“infusion materials”) into another gaseous or liquidmaterial (“host material”) reproduced from U.S. Pat. No. 6,386,751,incorporated herein by reference in its entirety. The device 10 includesa housing configured to house a stator 30 and a rotor 12. The stator 30encompasses the rotor 12. A tubular channel 32 is defined between therotor 12 and the stator 30. The generally cylindrically shaped rotor 12has a diameter of about 7.500 inches and a length of about 6.000 inchesproviding a length to diameter ratio of about 0.8.

The rotor 12 includes a hollow cylinder, generally closed at both ends.A gap exists between each of the first and second ends of the rotor 12and a portion of the housing 34. A rotating shaft 14 driven by a motor18 is coupled to the second end of the rotor 12. The first end of therotor 12 is coupled to an inlet 16. A first infusion material passesthrough the inlet 16 and into the interior of the rotor 12. The firstinfusion material passes from the interior of the rotor 12 and into thechannel 32 through a plurality of openings 22 formed in the rotor 12.

The stator 30 also has openings 22 formed about its circumference. Aninlet 36 passes a second infusion material to an area 35 between thestator 30 and the housing 34. The second infusion material passes out ofthe area 35 and into the channel 32 through openings 22.

An external pump (not shown) is used to pump the host material into asingle inlet port 37. The host material passes through a single inletport 37 and into the channel 32 where it encounters the first and secondinfusion materials, which enter the channel 32 through openings 22. Theinfusion materials may be pressurized at their source to prevent thehost material from passing through openings 22.

The inlet port 37, is configured and positioned such that it is locatedalong only a relatively small portion (<about 5%) of the annular inletchannel 32, and is substantially parallel to the axis of rotation of therotor 12 to impart an axial flow toward a portion of the channel 32 intothe host material.

Unfortunately, before entering the tubular channel 32, the host materialmust travel in tortuous directions other than that of the axial flow(e.g., including in directions substantially orthogonal thereto) anddown into and between the gap formed between the first end of the rotor12 and the housing 34 (i.e., down a portion of the first end of therotor adjacent to the inlet 16 between the end of the rotor 12 and thehousing 34). The non-axial and orthogonal flow, and the presence of thehost material in the gap between the first end of the rotor 12 and thehousing 34 causes undesirable and unnecessary friction. Further, it ispossible for a portion of the host material to become trapped in eddycurrents swirling between the first end of the rotor and the housing.Additionally, in the device 10, the host material must negotiate atleast two right angles to enter any aspect of the annual of the annularinlet of the tubular channel 32.

A single outlet port 40 is formed in the housing 34. The combined hostmaterial and infusion material(s) exit the channel 32 via the outlet 40.The outlet port 40, which is also located along only a limited portion(<about 5%) of the annular outlet of tubular channel 32, issubstantially parallel to the axis of rotation of the rotor 12 to impartor allow for an axial flow of the combined materials away from thelimited portion of the annular outlet of tubular channel 32 into theoutlet port 40. An external pump 42 is used to pump the exiting fluidthrough the outlet port 40.

Unfortunately, before exiting the channel 32, a substantial portion ofthe exiting material must travel in a tortuous direction other than thatof the axial flow (e.g., including in directions substantiallyorthogonal thereto) and down into and between the gap formed between thesecond end of the rotor 12 and the housing 34 (i.e., down a portion ofthe second end of the rotor adjacent to the shaft 14 between the end ofthe rotor 12 and the housing 34). As mentioned above, the non-axial andorthogonal flow, and the presence of the host material in the other gapbetween the end (in this case, the second end) of the rotor 12 and thehousing 34 causes additional undesirable and unnecessary friction.Further, it is possible for a portion of the host material to becometrapped in eddy currents swirling between the second end of the rotorand the housing. Additionally, in the device 10, a substantial portionof the exiting combined material must negotiate at least two rightangles as it exits form the annular exit of the tubular channel 32 intothe outlet port 40.

As is apparent to those of ordinary skill in the art, the inlet port 37imparts only an axial flow to the host material. Only the rotor 21imparts a circumferential flow into the host material. Further, theoutlet port 40 imparts or provides for only an axial flow into theexiting material. Additionally, the circumferential flow velocity vectoris imparted to the material only after it enters the annular inlet 37 ofthe tubular channel 32, and subsequently the circumferential flow vectormust be degraded or eliminated as the material enters the exit port 40.There is, therefore, a need for a progressive circumferentialacceleration of the material as it passes in the axial direction throughthe channel 32, and a circumferential deceleration upon exit of thematerial from the channel 32. These aspects, in combination with thetortuous path that the material takes from the inlet port 37 to theoutlet port 40, create a substantial friction and flow resistance overthe path that is accompanied by a substantial pressure differential (26psi, at 60 gallons/min flow rate) between the inlet 37 and outlet 40ports, and these factors, inter alia, combine to reduce the overallefficiency of the system.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWING(S)

FIG. 1 is a partial cross-section, partial block diagram of a prior artmixing device.

FIG. 2 is block diagram of an exemplary embodiment of a mixing device.

FIG. 3 is an illustration of an exemplary system for delivering a firstmaterial to the mixing device of FIG. 2.

FIG. 4 is a fragmentary partial cross-sectional view of a top portion ofthe mixing device of FIG. 2.

FIG. 5 is a fragmentary cross-sectional view of a first side portion ofthe mixing device of FIG. 2.

FIG. 6 is a fragmentary cross-sectional view of a second side portion ofthe mixing device of FIG. 2.

FIG. 7 is a fragmentary cross-sectional view of a side portion of themixing device of FIG. 2 located between the first side portion of FIG. 5and the second side portion of FIG. 6.

FIG. 8 is a perspective view of a rotor and a stator of the mixingdevice of FIG. 2.

FIG. 9 is a perspective view of an inside of a first chamber of themixing device of FIG. 2.

FIG. 10 is a fragmentary cross-sectional view of the inside of a firstchamber of the mixing device of FIG. 2 including an alternate embodimentof the pump 410.

FIG. 11 is a perspective view of an inside of a second chamber of themixing device of FIG. 2.

FIG. 12 is a fragmentary cross-sectional view of a side portion of analternate embodiment of the mixing device.

FIG. 13 is a perspective view of an alternate embodiment of a centralsection of the housing for use with an alternate embodiment of themixing device.

FIG. 14 is a fragmentary cross-sectional view of an alternate embodimentof a bearing housing for use with an alternate embodiment of the mixingdevice.

FIG. 15 is a cross-sectional view of the mixing chamber of the mixingdevice of FIG. 2 taken through a plane orthogonal to the axis ofrotation depicting a rotary flow pattern caused by cavitation bubbleswhen a through-hole of the rotor approaches (but is not aligned with) anaperture of the stator.

FIG. 16 is a cross-sectional view of the mixing chamber of the mixingdevice of FIG. 2 taken through a plane orthogonal to the axis ofrotation depicting a rotary flow pattern caused by cavitation bubbleswhen the through-hole of the rotor is aligned with the aperture of thestator.

FIG. 17 is a cross-sectional view of the mixing chamber of the mixingdevice of FIG. 2 taken through a plane orthogonal to the axis ofrotation depicting a rotary flow pattern caused by cavitation bubbleswhen a through-hole of the rotor that was previously aligned with theaperture of the stator is no longer aligned therewith.

FIG. 18 is a side view of an alternate embodiment of a rotor.

FIG. 19 is an enlarged fragmentary cross-sectional view taken through aplane orthogonal to an axis of rotation of the rotor depicting analternate configuration of through-holes formed in the rotor andthrough-holes formed in the stator.

FIG. 20 is an enlarged fragmentary cross-sectional view taken through aplane passing through and extending along the axis of rotation of therotor depicting a configuration of through-holes formed in the rotor andthrough-holes formed in the stator.

FIG. 21 is an enlarged fragmentary cross-sectional view taken through aplane passing through and extending along the axis of rotation of therotor depicting an alternate offset configuration of through-holesformed in the rotor and through-holes formed in the stator.

FIG. 22 is an illustration of a shape that may be used to construct thethrough-holes of the rotor and/or the apertures of the stator.

FIG. 23 is an illustration of a shape that may be used to construct thethrough-holes of the rotor and/or the apertures of the stator.

FIG. 24 is an illustration of a shape that may be used to construct thethrough-holes of the rotor and/or the apertures of the stator.

FIG. 25 is an illustration of a shape that may be used to construct thethrough-holes of the rotor and/or the apertures of the stator.

FIG. 26 is an illustration of an electrical double layer (“EDL”) formednear a surface.

FIG. 27 is a perspective view of a model of the inside of the mixingchamber.

FIG. 28 is a cross-sectional view of the model of FIG. 27.

FIG. 29 is an illustration of an experimental setup.

FIG. 30 illustrates dissolved oxygen levels in water processed withoxygen in the mixing device of FIG. 2 and stored a 500 ml thin walledplastic bottle and a 1,000 ml glass bottle each capped at 65°Fahrenheit.

FIG. 31 illustrates dissolved oxygen levels in water processed withoxygen in the mixing device of FIG. 2 and stored in a 500 ml plasticthin walled bottle and a 1,000 ml glass bottle both refrigerated at 39°Fahrenheit.

FIG. 32 illustrates the dissolved oxygen levels in GATORADE® processedwith oxygen in the mixing device of FIG. 2 and stored in 32 oz.GATORADE® bottles having an average temperature of 55° Fahrenheit.

FIG. 33 illustrates the dissolved oxygen retention of a 500 ml braunbalanced salt solution processed with oxygen in the mixing device ofFIG. 2.

FIG. 34 illustrates a further experiment wherein the mixing device ofFIG. 2 is used to sparge oxygen from water by processing the water withnitrogen in the mixing device of FIG. 2.

FIG. 35 illustrates the sparging of oxygen from water by the mixingdevice of FIG. 2 at standard temperature and pressure.

FIG. 36 is an illustration of a nanocage.

FIG. 37 illustrates the Rayleigh scattering effects produced by a sampleof the water processed with oxygen by the mixing device of FIG. 2.

FIGS. 38-41 illustrate the inventive oxygen-enriched fluid testedpositive for reactivity with horseradish peroxidase by pyrogallol, whilethe pressure pot and fine bubbled water samples had far less reactivity.

FIG. 42 illustrates pyrogallol/HRP assays as described herein, showingthat oxygen is required for the reaction with pyrogallol in the presenceof horseradish peroxidase, as inventive fluid enriched with other gases(argon and nitrogen) did not react in the same manner.

FIG. 43 illustrates the hydrogen peroxide positive control showed astrong reactivity, while none of the other fluids tested reacted withthe glutathione.

FIG. 44 illustrates T7 DNA shows a confirmational change at about 50degrees Celsius in the control (deionized water), whereas the DNA in theoxygen-enriched inventive fluid remains intact until about 60 degreesCelsius.

FIGS. 45A and 45B illustrate a graphical representation of a exemplaryembodiments of a bioreactor system 3300 a.

FIG. 46 shows detailed portions of exemplary embodiments of thebioreactor system 3300 a of FIGS. 45A and 45B.

SUMMARY OF EXEMPLARY EMBODIMENTS

Particular aspects provide a composition, comprising anelectrokinetically altered oxygenated aqueous fluid or solution, whereinthe oxygen in the fluid or solution is present in an amount of at least25 ppm, at least 30, at least 40, at least 50, or at least 60 ppmoxygen. In particular embodiments, the electrokinetically alteredoxygenated aqueous fluid or solution comprises electrokineticallymodified or charged oxygen species. In certain aspects, theelectrokinetically modified or charged oxygen species are present in anamount of at least 0.5 ppm, at least 1 ppm, at least 3 ppm, at least 5ppm, at least 7 ppm, at least 10 ppm, at least 15 ppm, or at least 20ppm. In particular embodiments, the electrokinetically alteredoxygenated aqueous fluid or solution comprises solvated electronsstabilized by molecular oxygen. In certain aspects, the solvatedelectrons are present in an amount of at least 0.01 ppm, at least 0.1ppm, at least 0.5 ppm, at least 1 ppm, at least 3 ppm, at least 5 ppm,at least 7 ppm, at least 10 ppm, at least 15 ppm, or at least 20 ppm. Inparticular embodiments, the fluid or solution facilitates oxidation ofpyrogallol to purpurogallin in the presence of horseradish peroxidaseenzyme (HRP) in an amount above that afforded by a control pressure potgenerated or fine-bubble generated aqueous fluid or solution having anequivalent dissolved oxygen level, and wherein there is no hydrogenperoxide, or less than 0.1 ppm of hydrogen peroxide present in theelectrokinetic oxygen-enriched aqueous fluid or solution. In certainaspects, the facilitation of oxidation of pyrogallol to purpurogallinpersists for at least three hours in an open container, or for at leasttwo months in a closed gas-tight container.

Additional aspects provide a composition, comprising anelectrokinetically altered oxygenated aqueous fluid or solution, whereinthe fluid or solution comprises at least 25 ppm, at least 30, at least40, at least 50, or at least 60 ppm oxygen, wherein the fluid orsolution facilitates oxidation of pyrogallol to purpurogallin in thepresence of horseradish peroxidase enzyme (HRP) in an amount above thatafforded by a control pressure pot generated or fine-bubble generatedaqueous fluid or solution having an equivalent dissolved oxygen level,and wherein there is no hydrogen peroxide, or less than 0.1 ppm ofhydrogen peroxide present in the electrokenetic oxygen-enriched aqueousfluid or solution. In particular embodiments, the facilitation ofoxidation of pyrogallol to purpurogallin persists for at least threehours in an open container, or for at least two months in a closedgas-tight container. In certain aspects, the oxygenated aqueous fluid orsolution comprises solvated electrons stabilized by molecular oxygen. Inparticular embodiments, the solvated electrons are present in an amountof at least 0.01 ppm, at least 0.1 ppm, at least 0.5 ppm, at least 1ppm, at least 3 ppm, at least 5 ppm, at least 7 ppm, at least 10 ppm, atleast 15 ppm, or at least 20 ppm.

Further aspects provide a method of producing an electrokineticallyaltered oxygenated aqueous fluid or solution, comprising: providing aflow of a fluid material between two spaced surfaces in relative motionand defining a mixing volume therebetween, wherein the dwell time of asingle pass of the flowing fluid material within and through the mixingvolume is greater than 0.06 seconds or greater than 0.1 seconds; andintroducing oxygen (O₂) into the flowing fluid material within themixing volume under conditions suitable to dissolve at least 20 ppm, atleast 25 ppm, at least 30, at least 40, at least 50, or at least 60 ppmoxygen into the material, and electrokinetically alter the fluid orsolution. In certain aspects, the oxygen is infused into the material inless than 100 milliseconds, less than 200 milliseconds, less than 300milliseconds, or less than 400 milliseconds. In particular embodiments,the ratio of surface area to the volume is at least 12, at least 20, atleast 30, at least 40, or at least 50.

Yet further aspects, provide a method of producing an electrokineticallyaltered oxygenated aqueous fluid or solution, comprising: providing aflow of a fluid material between two spaced surfaces defining a mixingvolume therebetween; and introducing oxygen into the flowing materialwithin the mixing volume under conditions suitable to infuse at least 20ppm, at least 25 ppm, at least 30, at least 40, at least 50, or at least60 ppm oxygen into the material in less than 100 milliseconds, less than200 milliseconds, less than 300 milliseconds, or less than 400milliseconds. In certain aspects, the dwell time of the flowing materialwithin the mixing volume is greater than 0.06 seconds or greater than0.1 seconds. In particular embodiments, the ratio of surface area to thevolume is at least 12, at least 20, at least 30, at least 40, or atleast 50.

Additional embodiments provide a method of producing anelectrokinetically altered oxygenated aqueous fluid or solution,comprising use of a mixing device for creating an output mixture bymixing a first material and a second material, the device comprising: afirst chamber configured to receive the first material from a source ofthe first material; a stator; a rotor having an axis of rotation, therotor being disposed inside the stator and configured to rotate aboutthe axis of rotation therein, at least one of the rotor and statorhaving a plurality of through-holes; a mixing chamber defined betweenthe rotor and the stator, the mixing chamber being in fluidcommunication with the first chamber and configured to receive the firstmaterial therefrom, and the second material being provided to the mixingchamber via the plurality of through-holes formed in the one of therotor and stator; a second chamber in fluid communication with themixing chamber and configured to receive the output material therefrom;and a first internal pump housed inside the first chamber, the firstinternal pump being configured to pump the first material from the firstchamber into the mixing chamber. In certain aspects, the first internalpump is configured to impart a circumferential velocity into the firstmaterial before it enters the mixing chamber.

Further embodiments provide a method of producing an electrokineticallyaltered oxygenated aqueous fluid or solution, comprising use of a mixingdevice for creating an output mixture by mixing a first material and asecond material, the device comprising: a stator; a rotor having an axisof rotation, the rotor being disposed inside the stator and configuredto rotate about the axis of rotation therein; a mixing chamber definedbetween the rotor and the stator, the mixing chamber having an openfirst end through which the first material enters the mixing chamber andan open second end through which the output material exits the mixingchamber, the second material entering the mixing chamber through atleast one of the rotor and the stator; a first chamber in communicationwith at least a majority portion of the open first end of the mixingchamber; and a second chamber in communication with the open second endof the mixing chamber.

Additional aspects provide an electrokinetically altered oxygenatedaqueous fluid or solution made according to any of the above methods.

DETAILED DESCRIPTION OF THE INVENTION Overview

FIG. 2 provides a block diagram illustrating some of the components of amixing device 100 and the flow of material into, within, and out of thedevice. The mixing device 100 combines two or more input materials toform an output material 102, which may be received therefrom into astorage vessel 104. The mixing device 100 agitates the two or more inputmaterials in a novel manner to produce an output material 102 havingnovel characteristics. The output material 102 may include not only asuspension of at least one of the input materials in at least one of theother input materials (e.g., emulsions) but also a novel combination(e.g., electrostatic combinations) of the input materials, a chemicalcompound resulting from chemical reactions between the input materials,combinations having novel electrostatic characteristics, andcombinations thereof.

The input materials may include a first material 110 provided by asource 112 of the first material, a second material 120 provided by asource 122 of the second material, and optionally a third material 130provided by a source 132 of the third material. The first material 110may include a liquid, such as water, saline solution, chemicalsuspensions, polar liquids, non-polar liquids, colloidal suspensions,cell growing media, and the like. In some embodiments, the firstmaterial 110 may include the output material 102 cycled back into themixing device 100. The second material 120 may consist of or include agas, such as oxygen, nitrogen, carbon dioxide, carbon monoxide, ozone,sulfur gas, nitrous oxide, nitric oxide, argon, helium, bromine, andcombinations thereof, and the like. In preferred embodiments, the gas isor comprises oxygen. The optional third material 130 may include eithera liquid or a gas. In some embodiments, the third material 130 may be orinclude the output material 102 cycled back into the mixing device 100(e.g., to one or more of the pumps 210, 220 or 230, and/or into thechamber 310, and/or 330).

Optionally, the first material 110, the second material 120, and theoptional third material 130 may be pumped into the mixing device 100 byan external pump 210, an external pump 220, and an external pump 230,respectively. Alternatively, one or more of the first material 110, thesecond material 120, and the optional third material 130 may be storedunder pressure in the source 112, the source 122, and the source 132,respectively, and may be forced into the mixing device 100 by thepressure. The invention is not limited by the method used to transferthe first material 110, the second material 120, and optionally, thethird material 130 into the mixing device 100 from the source 112, thesource 122, and the source 132, respectively.

The mixing device 100 includes a first chamber 310 and a second chamber320 flanking a mixing chamber 330. The three chambers 310, 320, and 330are interconnected and form a continuous volume.

The first material 110 is transferred into the first chamber 310 andflows therefrom into the mixing chamber 330. The first material 110 inthe first chamber 310 may be pumped into the first chamber 310 by aninternal pump 410. The second material 120 is transferred into themixing chamber 330. Optionally, the third material 130 may betransferred into the mixing chamber 330. The materials in the mixingchamber 330 are mixed therein to form the output material 102. Then, theoutput material 102 flows into the second chamber 320 from which theoutput material 102 exits the mixing device 100. The output material 102in the mixing chamber 330 may be pumped into the second chamber 320 byan internal pump 420. Optionally, the output material 102 in the secondchamber 320 may be pumped therefrom into the storage vessel 104 by anexternal pump 430 (e.g., alone or in combination with the internal pump410 and/or 420).

In particular aspects, a common drive shaft 500 powers both the internalpump 410 and the internal pump 420. The drive shaft 500 passes throughthe mixing chamber 330 and provides rotational force therein that isused to mix the first material 110, the second material 120, andoptionally, the third material 130 together. The drive shaft 500 ispowered by a motor 510 coupled thereto.

FIG. 3 provides a system 512 for supplying the first material 110 to themixing device 100 and removing the output material 102 from the mixingdevice 100. In the system 512, the storage vessel 104 of the outputmaterial 102 and the source 112 of the first material 110 are combined.The external pump 210 is coupled to the combined storage vessel 104 andsource 112 by a fluid conduit 514 such as hose, pipe, and the like. Theexternal pump 210 pumps the combined first material 110 and outputmaterial 102 from the combined storage vessel 104 and source 112 throughthe fluid conduit 514 and into a fluid conduit 516 connecting theexternal pump 210 to the mixing device 100. The output material 102exits the mixing device 100 through a fluid conduit 518. The fluidconduit 518 is coupled to the combined storage vessel 104 and source 112and transports the output material 102 exiting the mixing device 100 tothe combined storage vessel 104 and source 112. The fluid conduit 518includes a valve 519 that establishes an operating pressure or backpressure within the mixing device 100.

Referring to FIGS. 2, 4-10, and 11, a more detailed description ofvarious components of an embodiment of the mixing device 100 will beprovided. The mixing device 100 is scalable. Therefore, dimensionsprovided with respect to various components may be used to construct anembodiment of the device or may be scaled to construct a mixing deviceof a selected size.

Turning to FIG. 4, the mixing device 100 includes a housing 520 thathouses each of the first chamber 310, the mixing chamber 330, and thesecond chamber 320. As mentioned above, the mixing device 100 includesthe drive shaft 500, which rotates during operation of the device.Therefore, the mixing device 100 may vibrate or otherwise move.Optionally, the mixing device 100 may be coupled to a base 106, whichmay be affixed to a surface such as the floor to maintain the mixingdevice 100 in a substantially stationary position.

The housing 520 may be assembled from two or more housing sections. Byway of example, the housing 520 may include a central section 522flanked by a first mechanical seal housing 524 and a second mechanicalseal housing 526. A bearing housing 530 may be coupled to the firstmechanical seal housing 524 opposite the central section 522. A bearinghousing 532 may be coupled to the second mechanical seal housing 526opposite the central section 522. Optionally, a housing section 550 maybe coupled to the bearing housings 530.

Each of the bearing housings 530 and 532 may house a bearing assembly540 (see FIGS. 5 and 6). The bearing assembly 540 may include anysuitable bearing assembly known in the art including a model number“202SZZST” manufactured by SKF USA Inc, of Kulpsville, Pa., operating awebsite at www.skf.com.

Seals may be provided between adjacent housing sections. For example,o-ring 560 (see FIG. 5) may be disposed between the housing section 550and the bearing housing 530, o-ring 562 (see FIG. 5) may be disposedbetween the first mechanical seal housing 524 and the central section522, and o-ring 564 (see FIG. 6) may be disposed between the secondmechanical seal housing 526 and the central section 522.

Mixing Chamber 330

Turning now to FIG. 7, the mixing chamber 330 is disposed inside thecentral section 522 of the housing 520 between the first mechanical sealhousing 524 and the second mechanical seal housing 526. The mixingchamber 330 is formed between two components of the mixing device 100, arotor 600 and a stator 700. The rotor 600 may have a sidewall 604 withan inside surface 605 defining a generally hollow inside portion 610 andan outside surface 606. The sidewall 604 may be about 0.20 inches toabout 0.75 inches thick. In some embodiments, the sidewall 604 is about0.25 inches thick. However, because the mixing device 100 may be scaledto suit a particular application, embodiments of the device having asidewall 604 that is thicker or thinner than the values provided arewithin the scope of the present teachings. The sidewall 604 includes afirst end portion 612 and a second end portion 614 and a plurality ofthrough-holes 608 formed between the first end portion 612 and thesecond end portion 614. Optionally, the outside surface 606 of thesidewall 604 may include other features such as apertures, projections,textures, and the like. The first end portion 612 has a relieved portion616 configured to receive a collar 618 and the second end portion 614has a relieved portion 620 configured to receive a collar 622.

The rotor 600 is disposed inside the stator 700. The stator 700 has asidewall 704 with an inside surface 705 defining a generally hollowinside portion 710 into which the rotor 600 is disposed. The sidewall704 may be about 0.1 inches to about 0.3 inches thick. In someembodiments, the sidewall 604 is about 1.5 inches thick. The stator 700may be non-rotatably coupled to the housing 520 in a substantiallystationary position. Alternatively, the stator 700 may integrally formedwith the housing 520. The sidewall 704 has a first end portion 712 and asecond end portion 714. Optionally, a plurality of apertures 708 areformed in the sidewall 704 of the stator 700 between the first endportion 712 and the second end portion 714. Optionally, the insidesurface 705 of the sidewall 704 may include other features such asthrough-holes, projections, textures, and the like.

The rotor 600 rotates with respect to the stationary stator 700 about anaxis of rotation “α” in a direction indicated by arrow “C3” in FIG. 9.Each of the rotor 600 and the stator 700 may be generally cylindrical inshape and have a longitudinal axis. The rotor 600 has an outer diameter“D1” and the stator 700 may have an inner diameter “D2.” The diameter“D1” may range, for example, from about 0.5 inches to about 24 inches.In some embodiments, the diameter “D1” is about 3.04 inches. In someembodiments, the diameter “D1” is about 1.7 inches. The diameter “D2,”which is larger than the diameter “D1,” may range from about 0.56 inchesto about 24.25 inches.

In some embodiments, the diameter “D2” is about 4 inches. Therefore, themixing chamber 330 may have a ring-shaped cross-sectional shape that isabout 0.02 inches to about 0.125 inches thick (i.e., the differencebetween the diameter “D2” and the diameter “D1”). In particularembodiments, the mixing chamber 330 is about 0.025 inches thick. Thechannel 32 between the rotor 12 and the stator 34 of prior art device 10(see FIG. 1) has a ring-shaped cross-sectional shape that is about 0.09inches thick. Therefore, in particular embodiments, the thickness of themixing chamber 330 is less than about one third of the channel 32 of theprior art device 10.

The longitudinal axis of the rotor 600 may be aligned with its axis ofrotation “α.” The longitudinal axis of the rotor 600 may be aligned withthe longitudinal axis of the stator 700. The rotor 600 may have a lengthof about 3 inches to about 6 inches along the axis of rotation “α.” Insome embodiments, the rotor 600 may have a length of about 5 inchesalong the axis of rotation “α.” The stator 700 may have a length ofabout 3 inches to about 6 inches along the axis of rotation “α.” In someembodiments, the stator 700 may have a length of about 5 inches alongthe axis of rotation “α.”

While the rotor 600 and the stator 700 have been depicted as having agenerally cylindrical shape, those of ordinary skill in the artappreciate that alternate shapes may be used. For example, the rotor 600and the stator 700 may be conically, spherically, arbitrarily shaped,and the like. Further, the rotor 600 and the stator 700 need not beidentically shaped. For example, the rotor 600 may be cylindricallyshaped and the stator 700 rectangular shaped or vise versa.

The apertures 708 of the stator 700 and the through-holes 608 depictedin FIGS. 4-7 are generally cylindrically shaped. The diameter of thethrough-holes 608 may range from about 0.1 inches to about 0.625 inches.The diameter of the apertures 708 may range from about 0.1 inches toabout 0.625 inches. One or more of apertures 708 of the stator 700 mayhave a diameter that differs from the diameters of the other apertures708. For example, the apertures 708 may increase in diameter from thefirst end portion 712 of the stator 700 to the second end portion 714 ofthe stator 700, the apertures 708 may decrease in diameter from thefirst end portion 712 of the stator 700 to the second end portion 714 ofthe stator 700, or the diameters of the apertures 708 may vary inanother manner along the stator 700. One or more of through-holes 608 ofthe rotor 600 may have a diameter that differs from the diameters of theother through-holes 608. For example, the through-holes 608 may increasein diameter from the first end portion 612 of the rotor 600 to thesecond end portion 614 of the rotor 600, the through-holes 608 maydecrease in diameter from the first end portion 612 of the rotor 600 tothe second end portion 614 of the rotor 600, or the diameters of thethrough-holes 608 may vary in another manner along the rotor 600.

As described below with reference to alternate embodiments, theapertures 708 and the through-holes 608 may have shapes other thangenerally cylindrical and such embodiments are within the scope of thepresent invention. For example, the through-holes 608 may include anarrower portion, an arcuate portion, a tapered portion, and the like.Referring to FIG. 7, each of the through-holes 608 includes an outerportion 608A, a narrow portion 608B, and a tapered portion 608Cproviding a transition between the outer portion 608A and the narrowportion 608B. Similarly, the apertures 708 may include a narrowerportion, an arcuate portion, a tapered portion, and the like.

FIG. 8 provides a non-limiting example of a suitable arrangement of theapertures 708 of the stator 700 and the through-holes 608 of the rotor600. The apertures 708 of the stator 700 may be arranged insubstantially parallel lateral rows “SLAT-1” through “SLAT-6”substantially orthogonal to the axis of rotation “α.” The apertures 708of the stator 700 may also be arranged in substantially parallellongitudinal rows “SLONG-1” through “SLONG-7” substantially parallelwith the axis of rotation “α.” In other words, the apertures 708 of thestator 700 may be arranged in a grid-like pattern of orthogonal rows(i.e., the lateral rows are orthogonal to the longitudinal rows) havingthe longitudinal rows “SLONG-1” through “SLONG-7” substantially parallelwith the axis of rotation “α.”

Like the apertures 708 of the stator 700, the through-holes 608 of therotor 600 may be arranged in substantially parallel lateral rows“RLAT-1” through “RLAT-6” substantially orthogonal to the axis ofrotation “α.” However, instead of being arranged in a grid-like patternof orthogonal rows, the through-holes 608 of the rotor 600 may also bearranged in substantially parallel rows “RLONG-1” through “RLONG-7” thatextend longitudinally along a helically path. Alternatively, thethrough-holes 608 of the rotor 600 may also be arranged in substantiallyparallel rows “RLONG-1” through “RLONG-7” that extend longitudinally atan angle other than parallel with the axis of rotation “α.”

The apertures 708 of the stator 700 and the through-holes 608 of therotor 600 may be configured so that when the rotor 600 is disposedinside the stator 700 the lateral rows “SLAT-1” to “SLAT-6” at leastpartially align with the lateral rows “RLAT-1” to “RLAT-6,”respectively. In this manner, as the rotor 600 rotates inside the stator700, the through-holes 608 pass by the apertures 708.

The through-holes 608 in each of the lateral rows “RLAT-1” to “RLAT-6”may be spaced apart laterally such that all of the through-holes 608 inthe lateral row align, at least partially, with the apertures 708 in acorresponding one of the lateral rows “SLAT-1” to “SLAT-6” of the stator700 at the same time. The longitudinally extending rows “RLONG-1”through “RLONG-6” may be configured such that the through-holes 608 inthe first lateral row “RLAT-1” in each of the longitudinally extendingrows passes completely by the apertures 708 of the corresponding lateralrow “SLAT-1” before the through-holes 608 in the last lateral row“RLAT-6” begin to partially align with the apertures 708 of thecorresponding last lateral row “SLAT-6” of the stator 700.

While, in FIG. 8, six lateral rows and six longitudinally extending rowshave been illustrated with respect to the rotor 600 and six lateral rowsand seven longitudinally extending rows have been illustrated withrespect stator 700, it is apparent to those of ordinary skill in the artthat alternate numbers of lateral rows and/or longitudinal rows may beused with respect to the rotor 600 and/or stator 700 without departingfrom the present teachings.

To ensure that only one pair of openings between corresponding lateralrows will be coincident at any one time, the number of apertures 708 ineach of the lateral rows “SLAT-1” to “SLAT-6” on the stator 700 maydiffer by a predetermined number (e.g., one, two, and the like) thenumber of through-holes 608 in each of the corresponding lateral rows“RLAT-1” to “RLAT-6” on the rotor 600. Thus, for example, if lateral row“RLAT-1” has twenty through-holes 608 evenly spaced around thecircumference of rotor 600, the lateral row “SLAT-1” may have twentyapertures 708 evenly spaced around the circumference of stator 700.

Returning to FIG. 7, the mixing chamber 330 has an open first endportion 332 and an open second end portion 334. The through-holes 608formed in the sidewall 604 of the rotor 600 connect the inside portion610 of the rotor 600 with the mixing chamber 330.

The rotor 600 is rotated inside the stator 700 by the drive shaft 500aligned with the axis of rotation “α” of the rotor 600. The drive shaft500 may be coupled to the first end portion 612 and the second endportion 614 of the rotor 600 and extend through its hollow insideportion 610. In other words, a portion 720 of the drive shaft 500 isdisposed in the hollow inside portion 610 of the rotor 600.

The collar 618 is configured to receive a portion 721 of the drive shaft500 disposed in the hollow inside portion 610 and the collar 622 isconfigured to receive a portion 722 of the drive shaft 500 disposed inthe hollow inside portion 610.

The portion 721 has an outer diameter “D3” that may range from about 0.5inches to about 2.5 inches. In some embodiments, the diameter “D3” isabout 0.625 inches. The portion 722 has an outer diameter “D4” that maybe substantially similar to the diameter “D3,” although, this is notrequired. The diameter “D4” may range from about 0.375 inches to about2.5 inches.

The rotor 600 may be non-rotationally affixed to the portion 721 and theportion 722 of the drive shaft 500 by the collar 618 and the collar 622,respectively. By way of example, each of the collars 618 and 622 may beinstalled inside relieved portions 616 and 620, respectively. Then, thecombined rotor 600 and collars 618 and 622 may be heated to expand them.Next, the drive shaft 500 is inserted through the collars 618 and 622and the assembly is allowed to cool. As the collars 618 and 622 shrinkduring cooling, they tighten around the portions 722A and 722B of thedrive shaft 500, respectively, gripping it sufficiently tightly toprevent the drive shaft 500 from rotating relative to the rotor 600. Thecollar 618, which does not rotate with respect to either the portion 721or the relieved portion 616, translates the rotation of the drive shaft500 to the first end portion 612 the rotor 600. The collar 622, whichdoes not rotate with respect to either the portion 722 or the relievedportion 620, translates the rotation of the drive shaft 500 to thesecond end portion 614 of the rotor 600. The drive shaft 500 and therotor 600 rotate together as a single unit.

The drive shaft 500 may have a first end portion 724 (see FIG. 5) and asecond end portion 726 (see FIG. 6). The first end portion 724 may havea diameter “D5” of about 0.5 inches to about 1.75 inches. In particularembodiments, the diameter “D5” may be about 1.25 inches. The second endportion 726 may have a diameter “D6” that may be substantially similarto diameter “D5.”

The second material 120 may be transported into the mixing chamber 330through one of the first end portion 724 and the second end portion 726of the rotating drive shaft 500. The other of the first end portion 724and the second end portion 726 of the drive shaft 500 may be coupled tothe motor 510. In the embodiment depicted in FIGS. 5 and 6, the secondmaterial 120 is transported into the mixing chamber 330 through thefirst end portion 724 and the second end portion 726 of the drive shaft500 is coupled to the motor 510.

Turning to FIG. 5, the drive shaft 500 may have a channel 728 formedtherein that extends from first end portion 724 into the portion 720disposed in the inside portion 610 of the rotor 600. The channel 728 hasan opening 730 formed in the first end portion 724. When the mixingdevice 100 is operating, the second material 120 is introduced into thechannel 728 through the opening 730.

A valve 732 may be disposed inside a portion of the channel 728 locatedin the first end portion 724 of the drive shaft 500. The valve 732 mayrestrict or otherwise control the backward flow of the second material120 from inside the hollow inside portion 610 through the channel 728and/or the forward flow of the second material 120 into the channel 728.The valve 732 may include any valve known in the art including a checkvalve. A suitable check valve includes a part number “CKFA1876205A,”free flow forward check valve, manufactured by The Lee Company USAhaving an office in Bothell, Wash. and operating a website atwww.theleeco.com.

The drive shaft 500 may include an aperture 740 located in the insideportion 610 of the rotor 600 that connects the channel 728 with theinside portion 610 of the rotor 600. While only a single aperture 740 isillustrated in FIG. 5, it is apparent to those of ordinary skill in theart that multiple apertures may be used to connect the channel 728 withthe inside portion 610 of the rotor 600.

Referring to FIG. 2, optionally, the external pump 220 may pump thesecond material 120 into the mixing device 100. The pump 220 may includeany suitable pump known in the art. By way of non-limiting example, thepump 220 may include any suitable pump known in the art including adiaphragm pump, a chemical pump, a peristaltic pump, a gravity fed pump,a piston pump, a gear pump, a combination of any of the aforementionedpumps, and the like. If the second material 120 is a gas, the gas may bepressurized and forced into the opening 730 formed in the first endportion 724 of the drive shaft 500 by releasing the gas from the source122.

The pump 220 or the source 122 is coupled to the channel 728 by thevalve 732. The second material 120 transported inside the channel 728exits the channel 728 into the inside portion 610 of the rotor 600through the aperture 740. The second material 120 subsequently exits theinside portion 610 of the rotor 600 through the through-holes 608 formedin the sidewall 608 of the rotor 600.

Referring to FIG. 5, the mixing device 100 may include a seal assembly750 coupled to the first end portion 724 of the drive shaft 500. Theseal assembly 750 is maintained within a chamber 752 defined in thehousing 520. The chamber 752 has a first end portion 754 spaced acrossthe chamber from a second end portion 756. The chamber 752 also includesan input port 758 and an output port 759 that provide access into thechamber 752. The chamber 752 may be defined by housing section 550 andthe bearing housing 530. The first end portion 754 may be formed in thehousing section 550 and the second end portion 756 may be adjacent tothe bearing housing 530. The input port 758 may be formed in the bearinghousing 530 and the output port 759 may be formed in the housing section550.

The seal assembly 750 includes a first stationary seal 760 installed inthe first end portion 754 of the chamber 752 in the housing section 550and the bearing housing 530. The first stationary seal 760 extendsaround a portion 762 of the first end portion 724 of the drive shaft500. The seal assembly 750 also includes a second stationary seal 766installed in the second end portion 756 of the chamber 752 in thebearing housing 530. The second stationary seal 766 extends around aportion 768 of the first end portion 724 of the drive shaft 500.

The seal assembly 750 includes a rotating assembly 770 that isnon-rotatably coupled to the first end portion 724 of the drive shaft500 between the portion 762 and the portion 768. The rotating assembly770 rotates therewith as a unit. The rotating assembly 770 includes afirst seal 772 opposite a second seal 774. A biasing member 776 (e.g., aspring) is located between the first seal 772 and the second seal 774.The biasing member 776 biases the first seal 772 against the firststationary seal 760 and biases the second seal 774 against the secondstationary seal 766.

A cooling lubricant is supplied to the chamber 752 and around rotatingassembly 770. The lubricant enters the chamber 752 through the inputport 758 and exits the chamber 752 through output port 759. Thelubricant may lubricate the bearing assembly 540 housed by the bearinghousing 530. A chamber 570 may be disposed between the bearing housing530 and the mechanical seal housing 524. The bearing housing 530 mayalso include a second input port 759 connected to the chamber 570 intowhich lubricant may be pumped. Lubricant pumped into the chamber 570 maylubricate the bearing assembly 540. The seal assembly 750 maysignificantly, if not greatly, reduce frictional forces within thisportion of the device caused by the rotation of the rotor 600 and mayincrease the active life of the seals 770. The seals may includesurfaces constructed using silicon carbide.

Referring to FIG. 9, as the rotor 600 rotates about the axis of rotation“α” in the direction indicated by arrow “C1,” the rotor expels thesecond material 120 into the mixing chamber 330. The expelled bubbles,droplets, particles, and the like of the second material 120 exit therotor 600 and are imparted with a circumferential velocity (in adirection indicated by arrow “C3”) by the rotor 600. The second material120 may forced from the mixing chamber 330 by the pump 220 (see FIG. 2),the centrifugal force of the rotating rotor 600, buoyancy of the secondmaterial 120 relative to the first material 110, and a combinationthereof.

Motor 510

Returning to FIG. 6, the second end portion 726 of the drive shaft 500may be coupled to a rotating spindle 780 of a motor 510 by a coupler900. The spindle 780 may have a generally circular cross-sectional shapewith a diameter “D7” of about 0.25 inches to about 2.5 inches. Inparticular embodiments, the diameter “D7” may be about 0.25 inches toabout 1.5 inches. While in the embodiment depicted in FIG. 6, thediameter “D5” of the first end portion 724 of the drive shaft 500 issubstantially equal to the diameter “D7” and the spindle 780,embodiments in which one of the diameter “D5” and the diameter “D7” islarger than the other are within the scope of the present invention.

Referring also to FIG. 4, it may be desirable to cover or shield thecoupler 900. In the embodiment illustrated in FIGS. 4 and 6, a driveguard 910 covers the coupler 900. The drive guard 910 may be generallyU-shaped having a curved portion 914 flanked by a pair of substantiallylinear portions 915 and 916. The distal end of each of the substantiallylinear portions 915 and 916 of the drive guard 910 may have a flange 918and 919, respectively. The drive guard 910 may be fastened by each ofits flanges 918 and 919 to the base 106.

The motor 510 may be supported on the base 106 by a support member 920.The support member 920 may be coupled to the motor 510 near the spindle780. In the embodiment depicted, the support member 920 includes athrough-hole through which the spindle 780 passes. The support member920 may be coupled to the motor 510 using any method known in the art,including bolting the support member 920 to the motor 510 with one ormore bolts 940.

The coupler 900 may include any coupler suitable for transmitting asufficient amount of torque from the spindle 780 to the drive shaft 500to rotate the rotor 600 inside to the stator 700. In the embodimentillustrated in FIGS. 4 and 6, the coupler 900 is a bellows coupler. Abellows coupler may be beneficial if the spindle 780 and the drive shaft500 are misaligned. Further, the bellows coupler may help absorb axialforces exerted on the drive shaft 500 that would otherwise be translatedto the spindle 780. A suitable bellows coupler includes a model“BC32-8-8-A,” manufactured by Ruland Manufacturing Company, Inc. ofMarlborough, Mass., which operates a website at www.ruland.com.

The motor 510 may rotate the rotor 600 at about 0.1 revolutions perminute (“rpm”) to about 7200 rpm. The motor 510 may include any motorsuitable for rotating the rotor 600 inside to the stator 700 inaccordance with the present teachings. By way of non-limiting example, asuitable motor may include a one-half horsepower electric motor,operating at 230/460 volts and 3450 per minute (“rpm”). A suitable motorincludes a model “C4T34NC4C” manufactured by LEESON Electric Corporationof Grafton, Wis., which operates a website at www.leeson.com.

First Chamber 310

Turning to FIGS. 4 and 7, the first chamber 320 is disposed inside thecentral section 522 of the housing 520 between the first mechanical sealhousing 524 and the first end portions 612 and 712 of the rotor 600 andthe stator 700, respectively. The first chamber 310 may be annular andhave a substantially circular cross-sectional shape. The first chamber310 and the mixing chamber 330 form a continuous volume. A portion 1020of the drive shaft 500 extends through the first chamber 310.

As may best be viewed in FIG. 4, the first chamber 310 has an input port1010 through which the first material 110 enters the mixing device 100.The first material 110 may be pumped inside the first chamber 310 by theexternal pump 210 (see FIG. 2). The external pump 210 may include anypump known in the art for pumping the first material 110 at a sufficientrate to supply the first chamber 310.

The input port 1010 is oriented substantially orthogonally to the axisof rotation “α.” Therefore, the first material 110 enters the firstchamber 310 with a velocity tangential to the portion 1020 of the driveshaft 500 extending through the first chamber 310. The tangentialdirection of the flow of the first material 110 entering the firstchamber 310 is identified by arrow “T1.” In the embodiment depicted inFIGS. 4 and 7, the input port 1010 may be offset from the axis ofrotation “α.” As is apparent to those of ordinary skill in the art, thedirection of the rotation of the drive shaft 500 (identified by arrow“C1” in FIG. 9), has a tangential component. The input port 1010 ispositioned so that the first material 110 enters the first chamber 310traveling in substantially the same direction as the tangentialcomponent of the direction of rotation of the drive shaft 500.

The first material 110 enters the first chamber 310 and is deflected bythe inside of the first chamber 310 about the portion 1020 of the driveshaft 500. In embodiments wherein the first chamber 310 has asubstantially circular cross-sectional shape, the inside of the firstchamber 310 may deflect the first material 110 in a substantiallycircular path (identified by arrow “C2” in FIG. 9) about the portion1020 of the drive shaft 500. In such an embodiment, the tangentialvelocity of the first material 110 may cause it to travel about the axisof rotation “α” at a circumferential velocity, determined at least inpart by the tangential velocity.

Once inside the first chamber 310, the first material 110 may be pumpedfrom the first chamber 310 into the mixing chamber 330 by the pump 410residing inside the first chamber 310. In embodiments that include theexternal pump 210 (see FIG. 2), the external pump 210 may be configuredto pump the first material 110 into the first chamber 310 at a rate atleast as high as a rate at which the pump 410 pumps the first material110 from the first chamber 310.

The first chamber 310 is in communication with the open first endportion 332 of the mixing chamber 330 and the first material 110 insidethe first chamber 310 may flow freely into the open first end portion332 of the mixing chamber 330. In this manner, the first material 110does not negotiate any corners or bends between the mixing chamber 330and the first chamber 310. In the embodiment depicted, the first chamber310 is in communication with the entire open first end portion 332 ofthe mixing chamber 330. The first chamber 310 may be filled completelywith the first material 110.

The pump 410 is powered by the portion 1020 of the drive shaft 500extending through the first chamber 310. The pump 410 may include anypump known in the art having a rotating pump member 2022 housed inside achamber (i.e., the first chamber 310) defined by a stationary housing(i.e., the housing 520). Non-limiting examples of suitable pumps includerotary positive displacement pumps such as progressive cavity pumps,single screw pumps (e.g., Archimedes screw pump), and the like.

The pump 410 depicted in FIGS. 7 and 9, is generally referred to as asingle screw pump. In this embodiment, the pump member 2022 includes acollar portion 2030 disposed around the portion 1020 of the drive shaft500. The collar portion 2030 rotates with the portion 1020 of the driveshaft 500 as a unit. The collar portion 2030 includes one or more fluiddisplacement members 2040. In the embodiment depicted in FIGS. 7 and 9,the collar portion 2030 includes a single fluid displacement member 2040having a helical shape that circumscribes the collar portion 2030 alonga helical path.

Referring to FIG. 9, the inside of the first chamber 310 is illustrated.The pump 410 imparts an axial flow (identified by arrow “A1” and arrow“A2”) in the first material 110 inside the first chamber 310 toward theopen first end portion 332 of the mixing chamber 330. The axial flow ofthe first material 110 imparted by the pump 410 has a pressure that mayexceed the pressure obtainable by the external pump of the prior artdevice 10 (see FIG. 1).

The pump 410 may also be configured to impart a circumferential flow(identified by arrow “C2”) in the first material 110 as it travelstoward the open first end portion 332 of the mixing chamber 330. Thecircumferential flow imparted in the first material 110 before it entersthe mixing chamber 330 causes the first material 110 to enter the mixingchamber 330 already traveling in the desired direction at an initialcircumferential velocity. In the prior art device 10 depicted in FIG. 1,the first material 110 entered the channel 32 of the prior art device 10without a circumferential velocity. Therefore, the rotor 12 of the priorart device 10 alone had to impart a circumferential flow into the firstmaterial 110. Because the first material 110 is moving axially, in theprior art device 10, the first material 110 traversed at least a portionof the channel 32 formed between the rotor 12 and the stator 30 at aslower circumferential velocity than the first material 110 traversesthe mixing chamber 330 of the mixing device 100. In other words, if theaxial velocity of the first material 110 is the same in both the priorart device 10 and the mixing device 100, the first material 110 maycomplete more revolutions around the rotational axis “α” beforetraversing the axial length of the mixing chamber 330, than it wouldcomplete before traversing the axial length of the channel 32. Theadditional revolutions expose the first material 110 (and combined firstmaterial 110 and second material 120) to a substantially larger portionof the effective inside surface 706 (see FIG. 7) of the stator 700.

In embodiments including the external pump 210 (see FIG. 2), thecircumferential velocity imparted by the external pump 210 combined withthe input port 1010 being oriented according to the present teachings,may alone sufficiently increase the revolutions of the first material110 (and combined first material 110 and second material 120) about therotational axis “α.” Further, in some embodiments, the circumferentialvelocity imparted by the pump 210 and the circumferential velocityimparted by the pump 410 combine to achieve a sufficient number ofrevolutions of the first material 110 (and combined first material 110and second material 120) about the rotational axis “α.” As isappreciated by those of ordinary skill in the art, other structuralelements such as the cross-sectional shape of the first chamber 310 maycontribute to the circumferential velocity imparted by the pump 210, thepump 410, and a combination thereof.

In an alternate embodiment depicted in FIG. 10, the pump 410 may includeone or more vanes 2042 configured to impart a circumferential flow inthe first material 110 as it travels toward the open first end portion332 of the mixing chamber 330.

Second Chamber 320

Turning now to FIGS. 4 and 7, the second chamber 320 is disposed insidethe central section 522 of the housing 520 between the second mechanicalseal housing 526 and the second end portions 614 and 714 of the rotor600 and the stator 700, respectively. The second chamber 320 may besubstantially similar to the first chamber 310. However, instead of theinput port 1010, the second chamber 320 may include an output port 3010.A portion 3020 of the drive shaft 500 extends through the second chamber320.

The second chamber 320 and the mixing chamber 330 form a continuousvolume. Further, the first chamber 310, the mixing chamber 330, and thesecond chamber 320 form a continuous volume. The first material 110flows through the mixing device 100 from the first chamber 310 to themixing chamber 330 and finally to the second chamber 320. While in themixing chamber 330, the first material 110 is mixed with the secondmaterial 120 to form the output material 102. The output material 102exits the mixing device 100 through the output port 3010. Optionally,the output material 102 may be returned to the input port 1010 and mixedwith an additional quantity of the second material 120, the thirdmaterial 130, or a combination thereof.

The output port 3010 is oriented substantially orthogonally to the axisof rotation “α” and may be located opposite the input port 1010 formedin the first chamber 310. The output material 102 enters the secondchamber 320 from the mixing chamber 330 having a circumferentialvelocity (in the direction indicated by arrow “C3” in FIG. 9) impartedthereto by the rotor 600. The circumferential velocity is tangential tothe portion 3020 of the drive shaft 500 extending through the secondchamber 320. In the embodiment depicted in FIGS. 4, 6, and 7, the outputport 3010 may be offset from the axis of rotation “α.” The output port3010 is positioned so that the output material 102, which enters thesecond chamber 320 traveling in substantially the same direction inwhich the drive shaft 500 is rotating (identified in FIG. 9 by arrow“C1”), is traveling toward the output port 3010.

The output material 102 enters the second chamber 320 and is deflectedby the inside of the second chamber 320 about the portion 3020 of thedrive shaft 500. In embodiments wherein the second chamber 320 has asubstantially circular cross-sectional shape, the inside of the secondchamber 320 may deflect the output material 102 in a substantiallycircular path about the portion 3020 of the drive shaft 500.

Referring to FIG. 2, optionally, the output material 102 may be pumpedfrom inside the second chamber 320 by the external pump 430. Theexternal pump 430 may include any pump known in the art for pumping theoutput material 102 at a sufficient rate to avoid limiting throughput ofthe mixing device 100. In such an embodiment, the external pump 430 mayintroduce a tangential velocity (in a direction indicated by arrow “T2”in FIGS. 4 and 11) to at least a portion of the output material 102 asthe external pump 430 pumps the output material 102 from the secondchamber 320. The tangential velocity of the portion of the outputmaterial 102 may cause it to travel about the axis of rotation “α” at acircumferential velocity, determined in part by the tangential velocity.

Pump 420

Turning to FIGS. 6 and 7, the pump 420 residing inside the secondchamber 320 may pump the output material 102 from the second chamber 320into the output port 3010 and/or from the mixing chamber 330 into thesecond chamber 320. In embodiments that include the external pump 430,the external pump 430 may be configured to pump the output material 102from the second chamber 320 at a rate at least as high as a rate atwhich the pump 420 pumps the output material 102 into the output port3010.

The second chamber 320 is in communication with the open second endportion 334 of the mixing chamber 330 and the output material 102 insidethe mixing chamber 330 may flow freely from the open second end portion334 into the second chamber 320. In this manner, the output material 102does not negotiate any corners or bends between the mixing chamber 330and the second chamber 320. In the embodiment depicted, the secondchamber 320 is in communication with the entire open second end portion334 of the mixing chamber 330. The second chamber 320 may be filledcompletely with the output material 102.

The pump 420 is powered by the portion 3020 of the drive shaft 500extending through the second chamber 320. The pump 420 may besubstantially identical to the pump 410. Any pump described above assuitable for use as the pump 410 may be used for the pump 420. While thepump 410 pumps the first material 110 into the mixing chamber 330, thepump 420 pumps the output material 102 from the mixing chamber 330.Therefore, both the pump 410 and the pump 420 may be oriented to pump inthe same direction.

As is appreciated by those of ordinary skill in the art, the firstmaterial 110 may differ from the output material 102. For example, oneof the first material 110 and the output material 102 may be moreviscous than the other. Therefore, the pump 410 may differ from the pump420. The pump 410 may be configured to accommodate the properties of thefirst material 110 and the pump 420 may be configured to accommodate theproperties of the output material 102.

The pump 420 depicted in FIGS. 6 and 7, is generally referred to as asingle screw pump. In this embodiment, the pump member 4022 includes acollar portion 4030 disposed around the portion 3020 of the drive shaft500. The collar portion 4030 rotates with the portion 3020 of the driveshaft 500 as a unit. The collar portion 4030 includes one or more fluiddisplacement members 4040. The collar portion 4030 includes a singlefluid displacement member 4040 having a helical shape that circumscribesthe collar portion 4030 along a helical path.

Referring to FIG. 11, the inside of the second chamber 320 isillustrated. The pump 420 imparts an axial flow (identified by arrow“A3” and arrow “A4”) in the output material 102 inside the secondchamber 320 away from the open second end portion 334 of the mixingchamber 330.

The pump 420 may be configured to impart a circumferential flow(identified by arrow “C4”) in the output material 102 as it travels awayfrom the open second end portion 334 of the mixing chamber 330. Thecircumferential flow imparted in the output material 102 may help reducean amount of work required by the rotor 600. The circumferential flowalso directs the output material 102 toward the output port 3010.

In an alternate embodiment, the pump 420 may have substantially the sameconfiguration of the pump 410 depicted in FIG. 10. In such anembodiment, the one or more vanes 2042 are configured to impart acircumferential flow in the output material 102 as it travels away fromthe open second end portion 334 of the mixing chamber 330.

As is apparent to those of ordinary skill, various parameters of themixing device 100 may be modified to obtain different mixingcharacteristics. Exemplary parameters that may be modified include thesize of the through-holes 608, the shape of the through-holes 608, thearrangement of the through-holes 608, the number of through-holes 608,the size of the apertures 708, the shape of the apertures 708, thearrangement of the apertures 708, the number of apertures 708, the shapeof the rotor 600, the shape of the stator 700, the width of the mixingchamber 330, the length of the mixing chamber 330, rotational speed ofthe drive shaft 500, the axial velocity imparted by the internal pump410, the circumferential velocity imparted by the internal pump 410, theaxial velocity imparted by the internal pump 420, the circumferentialvelocity imparted by the internal pump 420, the configuration ofdisturbances (e.g., texture, projections, recesses, apertures, and thelike) formed on the outside surface 606 of the rotor 600, theconfiguration of disturbances (e.g., texture, projections, recesses,apertures, and the like) formed on the inside surface 706 of the stator700, and the like.

Alternate Embodiment

Referring to FIG. 12, a mixing device 5000 is depicted. The mixingdevice 5000 is an alternate embodiment of the mixing device 100.Identical reference numerals have been used herein to identifycomponents of the mixing device 5000 that are substantially similarcorresponding components of the mixing device 100. Only components ofthe mixing device 5000 that differ from the components of the mixingdevice 100 will be described.

The mixing device 5000 includes a housing 5500 for housing the rotor 600and the stator 5700. The stator 5700 may be non-rotatably couple by itsfirst end portion 5712 and its second end portion 5714 to the housing5500. A chamber 5800 is defined between the housing 5500 and a portion5820 of the stator 5700 flanked by the first end portion 5712 and thesecond end portion 5714. The housing 5500 includes an input port 5830which provides access into the chamber 5800. The input port 5830 may beoriented substantially orthogonally to the axis of rotation “α.”however, this is not a requirement.

The stator 5700 includes a plurality of through-holes 5708 that connectthe chamber 5800 and the mixing chamber 330 (defined between the rotor600 and the stator 5700). An external pump 230 may be used to pump thethird material 130 (which may be identical to the second material 120)into the chamber 5800 via the input port 5830. The third material 130pumped into the chamber 5800 may enter the mixing chamber 330 via thethrough-holes 5708 formed in the stator 5700. The third material 130 mayforced from the channel 5800 by the pump 230, buoyancy of the thirdmaterial 130 relative to the first material 110, and a combinationthereof. As the rotor 600 rotates, it may also draw the third material130 from the channel 5800 into the mixing chamber 330. The thirdmaterial 130 may enter the mixing chamber 330 as bubbles, droplets,particles, and the like, which are imparted with a circumferentialvelocity by the rotor 600.

Alternate Embodiment

An alternate embodiment of the mixing device 100 may be constructedusing a central section 5900 depicted in FIG. 13 and a bearing housing5920 depicted in FIG. 14. FIG. 13 depicts the central section 5900having in its interior the stator 700 (see FIG. 7). Identical referencenumerals have been used herein to identify components associated withthe central section 5900 that are substantially similar correspondingcomponents of the mixing device 100. Only components of the centralsection 5900 that differ from the components of the central section 522will be described. The central section 5900 and the stator 700 are bothconstructed from a conductive material such as a metal (e.g., stainlesssteel). The input port 1010 and the output port 3010 are bothconstructed from a nonconductive material such as plastic (e.g., PET,Teflon, nylon, PVC, polycarbonate, ABS, Delrin, polysulfone, etc.).

An electrical contact 5910 is coupled to the central section 5900 andconfigured to deliver a charge thereto. The central section 5900conducts an electrical charge applied to the electrical contact 5910 tothe stator 700. In further embodiments, the central section 5900 may beconstructed from a nonconductive material. In such embodiments, theelectrical contact 5910 may pass through the central section 5900 andcoupled to the stator 700. The electric charge applied by the electricalcontact 5910 to the stator 700 may help facilitate redox or otherchemical reactions inside the mixing chamber 330.

Optionally, insulation (not shown) may be disposed around the centralsection 5900 to electrically isolate it from the environment. Further,insulation may be used between the central section 5900 and the firstand second mechanical seals 524 and 526 that flank it to isolate itelectrically from the other components of the mixing device.

Turning now to FIG. 14, the bearing housing 5920 will be described. Thebearing housing 5920 is disposed circumferentially around the portion726 of the drive shaft 500. An electrical contact 5922 is coupled to thebearing housing 5920. A rotating brush contact 5924 provides anelectrical connection between the drive shaft 500 and the electricalcontact 5922.

In this embodiment, the drive shaft 500 and the rotor 600 are bothconstructed from a conductive material such as a metal (e.g., stainlesssteel). The bearing housing 5920 may be constructed from either aconductive or a nonconductive material. An electrical charge is appliedto the drive shaft 500 by the electrical contact 5922 and the rotatingbrush contact 5924. The electrical charge is conducted by the driveshaft 500 to the rotor 600.

The alternate embodiment of the mixing device 100 constructed using thecentral section 5900 depicted in FIG. 13 and the bearing housing 5920depicted in FIG. 14 may be operated in at least two ways. First, theelectrical contacts 5910 and 5922 may be configured not to provide anelectrical charge to the stator 700 and the rotor 600, respectively. Inother words, neither of the electrical contacts 5910 and 5922 areconnected to a current source, a voltage source, and the like.Alternatively, the electrical contacts 5910 and 5922 may be configuredto provide an electrical charge to the stator 700 and the rotor 600,respectively. For example, the electrical contacts 5910 and 5922 may becoupled to a DC voltage source (not shown) supplying a steady orconstant voltage across the electrical contacts 5910 and 5922. Thenegative terminal of the DC voltage source may be coupled to either ofthe electrical contacts 5910 and 5922 and the positive terminal of theDC voltage source may be coupled to the other of the electrical contacts5910 and 5922. The voltage supplied across the electrical contacts 5910and 5922 may range from about 0.0001 volts to about 1000 volts. Inparticular embodiments, the voltage may range from about 1.8 volts toabout 2.7 volts. By way of another example, a pulsed DC voltage having aduty cycle of between about 1% to about 99% may be used.

While the above examples of methods of operating the mixing device applya DC voltage across the electrical contacts 5910 and 5922, as isapparent to those of ordinary skill in the art, a symmetrical AC voltageor non symmetrical AC voltage having various shapes and magnitudes maybe applied across the electrical contacts 5910 and 5922 and suchembodiments are within the scope of the present invention.

Mixing Inside the Mixing Chamber 330

As mentioned above, in the prior art device 10 (shown in FIG. 1), thefirst material 110 entered the channel 32 between the rotor 12 and thestator 30 via a single limited input port 37 located along only aportion of the open second end of the channel 32. Likewise, the outputmaterial 102 exited the channel 32 via a single limited output port 40located along only a portion of the open first end of the channel 32.This arrangement caused undesirable and unnecessary friction. Byreplacing the single limited inlet port 37 and the single limited outletport 40 with the chambers 310 and 320, respectively, friction has beenreduced. Moreover, the first material 110 does not negotiate a cornerbefore entering the mixing chamber 330 and the output material 102 doesnot negotiate a corner before exiting the mixing chamber 330. Further,the chambers 310 and 320 provide for circumferential velocity of thematerial prior to entering, and after exiting the channel 32.

Accordingly, pressure drop across the mixing device 100 has beensubstantially reduced. In the embodiments depicted in FIGS. 2, 4-9, and11, the pressure drop between the input port 1010 and the output port3010 is only approximately 12 psi when the mixing device 100 isconfigured to produce about 60 gallons of the output material 102 perminute. This is an improvement over the prior art device 10 depicted inFIG. 1, which when producing about 60 gallons of output material perminute was at least 26 psi. In other words, the pressure drop across themixing device 100 is less than half that experienced by the prior artdevice 10.

According to additional aspects, the inclusion of pumps 410 and 420,which are powered by the drive shaft 500, provides a configuration thatis substantially more efficient in mixing materials and that requiresless energy than the external pumps used in the prior art.

Micro-Cavitation

During operation of the mixing device 100, the input materials mayinclude the first material 110 (e.g., a fluid) and the second material120 (e.g., a gas). The first material 110 and the second material 120are mixed inside the mixing chamber 330 formed between the rotor 600 andthe stator 700. Rotation of the rotor 600 inside the stator 700 agitatesthe first material 110 and the second material 120 inside the mixingchamber 330. The through-holes 608 formed in the rotor 600 and/or theapertures 708 formed in the stator 700 impart turbulence in the flow ofthe first material 110 and the second material 120 inside the mixingchamber 330.

Without being limited by theory, the efficiency and persistence of thediffusion of the second material 120 into the first material 110 isbelieved to be caused in part by micro-cavitation, which is described inconnection with FIGS. 15-17. Whenever a material flows over a smoothsurface, a rather laminar flow is established with a thin boundary layerthat is stationary or moving very slowly because of the surface tensionbetween the moving fluid and the stationary surface. The through-holes608 and optionally, the apertures 708, disrupt the laminar flow and cancause localized compression and decompression of the first material 110.If the pressure during the decompression cycle is low enough, voids(cavitation bubbles) will form in the material. The cavitation bubblesgenerate a rotary flow pattern 5990, like a tornado, because thelocalized area of low pressure draws the host material and the infusionmaterial, as shown in FIG. 15. When the cavitation bubbles implode,extremely high pressures result. As two aligned openings (e.g., one ofthe apertures 708 and one of the through-holes 608) pass one another, asuccussion (shock wave) occurs, generating significant energy. Theenergy associated with cavitation and succussion mixes the firstmaterial 110 and the second material 120 together to an extremely highdegree, perhaps at the molecular level.

The tangential velocity of the rotor 600 and the number of openings thatpass each other per rotation may dictate the frequency at which themixing device 100. It has been determined that operating the mixingdevice 100 within in the ultrasonic frequency range can be beneficial inmany applications. It is believed that operating the mixing device 100in the ultrasonic region of frequencies provides the maximum successionshock energy to shift the bonding angle of the fluid molecule, whichenables it to transport an additional quantity of the second material120 which it would not normally be able to retain. When the mixingdevice 100 is used as a diffuser, the frequency at which the mixingdevice 100 operates appears to affect the degree of diffusion, leadingto much longer persistence of the second material 120 (infusionmaterial) in the first material 110 (host material).

Referring now to FIG. 18, an alternate embodiment of the rotor 600,rotor 6000 is provided. The cavitations created within the firstmaterial 110 in the mixing chamber 330 may be configured to occur atdifferent frequencies along the length of the mixing chamber 330. Thefrequencies of the cavitations may be altered by altering the numberand/or the placement of the through-holes 6608 along the length of therotor 600. Each of the through-holes 6608 may be substantially similarto the through-holes 608 (discussed above).

By way of non-limiting example, the rotor 6000 may be subdivided intothree separate exemplary sections 6100, 6200, and 6300. Thethrough-holes 6608 increase in density from the section 6100 to thesection 6200, the number of holes in the section 6100 being greater thanthe number of holes in the section 6200. The through-holes 6608 alsoincrease in density from the section 6200 to the section 6300, thenumber of holes in the section 6200 being greater than the number ofholes in the section 6300. Each of the sections 6100, 6200, and 6300create succussions within their particular area at a different frequencydue to the differing numbers of through-holes 6608 formed therein.

By manufacturing the rotor 6000 with a desired number of through-holes6608 appropriately arranged in a particular area, the desired frequencyof the succussions within the mixing chamber 330 may be determined.Similarly, the desired frequency of the cavitations may be determined bya desired number of apertures 708 appropriately arranged in a particulararea upon the stator 700 within which the rotor 600 rotates. Further,the desired frequency (or frequencies) of the succussions within themixing chamber 330 may be achieved by selecting both a particular numberand arrangement of the apertures 708 formed in the stator 700 and aparticular number and arrangement of the through-holes 608 formed in therotor 600.

FIGS. 19-21, depict various alternative arrangements of the apertures708 formed in the stator 700 and the through-holes 608 formed in therotor 600 configured to achieve different results with respect to thecavitations created. FIG. 19 illustrates a configuration in which theapertures 708 and the through-holes 608 are aligned along an axis 7000that is not parallel with any line (e.g., line 7010) drawn through theaxis of rotation “α” of the rotor 600. In other words, if the rotor 600has a cylindrical shape, the axis 7000 does not pass through the centerof the rotor 600. Thus, the first material 110 within the mixing chamber330 will not be oriented perpendicularly to the compressions anddecompressions created by the apertures 708 and the through-holes 608.The compressions and decompressions will instead have a force vectorthat has at least a component parallel to the circumferential flow (inthe direction of arrow “C3” of FIG. 9) of first material 110 within themixing chamber 330.

Relative alignment of the apertures 708 and the through-holes 608 mayalso affect the creation of cavitations in the mixing chamber 330. FIG.20 illustrates an embodiment in which the apertures 708 are inregistration across the mixing chamber 330 with the through-holes 608.In this embodiment, rotation of the rotor 600 brings the through-holes608 of the rotor into direct alignment with the apertures 708 of thestator 700. When in direct alignment with each other, the compressiveand decompressive forces created by the apertures 708 and thethrough-holes 608 are directly aligned with one another.

In the embodiment depicted in FIG. 21, the apertures 708 and thethrough-holes 608 are offset by an offset amount “X” along the axis ofrotation “α.”. By way of non-limiting example, the offset amount “X” maybe determined as a function of the size of the apertures 708. Forexample, the offset amount “X” may be approximately equal to one half ofthe diameter of the apertures 708. Alternatively, the offset amount “X”may be determined as a function of the size of the through-holes 608.For example, the offset amount “X” may be approximately equal to onehalf of the diameter of the through-holes 608. If features (e.g.,recesses, projections, etc.) other than or in addition to thethrough-holes 608 and the apertures 708 are included in either the rotor600 or the stator 700, the offset amount “X” may be determined as afunction of the size of such features. In this manner, the compressiveand decompressive forces caused by the apertures 708 of the stator 700and the through-holes 608 of the rotor 600 collide at a slight offsetcausing additional rotational and torsional forces within the mixingchamber 330. These additional forces increase the mixing (e.g.,diffusive action) of the second material 120 into the first material 110within the mixing chamber 330.

Referring now to FIGS. 22-25, non-limiting examples of suitablecross-sectional shapes for the apertures 708 and the through-holes 608are provided. The cross-sectional shape of the apertures 708 and/or thethrough-holes 608 may be square as illustrated in FIG. 22, circular asillustrated in FIG. 23, and the like.

Various cross-sectional shapes of apertures 708 and/or the through-holes608 may be used to alter flow of the first material 110 as the rotor 600rotates within the stator 700. For example, FIG. 24 depicts a teardropcross-sectional shape having a narrow portion 7020 opposite a wideportion 7022. If the through-holes 608 have this teardrop shape, whenthe rotor 600 is rotated (in the direction generally indicated by thearrow “F”), the forces exerted on the first material 110, the secondmaterial 120, and optionally the third material 130 within the mixingchamber 330 increase as the materials pass from the wide portion 7022 ofthe teardrop to the narrow portion 7020.

Additional rotational forces can be introduced into the mixing chamber330 by forming the apertures 708 and/or the through-holes 608 with aspiral configuration as illustrated in FIG. 25. Material that flows intoand out of the apertures 708 and/or the through-holes 608 having thespiral configuration experience a rotational force induced by the spiralconfiguration. The examples illustrated in FIGS. 22-25 are provided asnon-limiting illustrations of alternate embodiments that may be employedwithin the mixing device 100. By application of ordinary skill in theart, the apertures 708 and/or the through-holes 608 may be configured innumerous ways to achieve various succussive and agitative forcesappropriate for mixing materials within the mixing chamber 330.

Double Layer Effect

The mixing device 100 may be configured to create the output material102 by complex and non-linear fluid dynamic interaction of the firstmaterial 110 and the second material 120 with complex, dynamicturbulence providing complex mixing that further favors electrokineticeffects (described below). The result of these electrokinetic effectsmay be observed within the output material 102 as charge redistributionsand redox reactions, including in the form of solvated electrons thatare stabilized within the output material.

Ionization or dissociation of surface groups and/or adsorption of ionsfrom a liquid cause most solid surfaces in contact with the liquid tobecome charged. Referring to FIG. 26, an electrical double layer (“EDL”)7100 forms around exemplary surface 7110 in contact with a liquid 7120.In the EDL 7100, ions 7122 of one charge (in this case, negativelycharged ions) adsorb to the surface 7120 and form a surface layer 7124typically referred to as a Stern layer. The surface layer 7124 attractscounterions 7126 (in this case, positively charged ions) of the oppositecharge and equal magnitude, which form a counterion layer 7128 below thesurface layer 7124 typically referred to as a diffuse layer. Thecounterion layer 7128 is more diffusely distributed than the surfacelayer 7124 and sits upon a uniform and equal distribution of both ionsin the bulk material 7130 below. For OH− and H+ ions in neutral water,the Gouy-Chapman model would suggest that the diffuse counterion layerextends about one micron into the water.

According to particular aspects, the electrokinetic effects mentionedabove are caused by the movement of the liquid 7120 next to the chargedsurface 7110. Within the liquid 7120 (e.g., water, saline solution, andthe like), the adsorbed ions 7122 forming the surface layer 7124 arefixed to the surface 7120 even when the liquid 7120 is in motion (forexample, flowing in the direction indicated by arrow “G”); however, ashearing plane 7132 exists within the diffuse counterion layer 7128spaced from the surface 7120. Thus, as the liquid 7120 moves, some ofthe diffuse counterions 7126 are transported away from the surface 7120,while the absorbed ions 7122 remain at the surface 7120. This produces aso-called ‘streaming current.’

Within the mixing chamber 330, the first material 110, the secondmaterial 120, and optionally, the third material 130 are subject to anelectromagnetic field created by the inside surface 705 of the stator700 and/or the outside surface 606 of the rotor 600, a voltage betweenthe inside surface 705 and the outside surface 606, and/or anelectrokinetic effect (e.g., streaming current) caused by at least oneEDL formed in the first material 110. The at least one EDL may beintroduced into the first material 110 by at least one of the insidesurface 705 of the stator 700 and the outside surface 606 of the rotor600.

Movement of the first material 110 through the mixing chamber 330relative to surface disturbances (e.g., the through-holes 608 andapertures 708) creates cavitations in the first material 110 within themixing chamber 330, which may diffuse the second material 120 into thefirst material 110. These cavitations may enhance contact between of thefirst material 110 and/or the second material 120 with the electricdouble layer formed on the inside surface 705 of the stator 700 and/orthe electric double layer formed on the outside surface 606 of the rotor600. Larger surface to volume ratios of the mixing chamber, an increaseddwell time of the combined materials within the mixing chamber, andfurther in combination with a smaller average bubble size (and hencesubstantially greater bubble surface area) provide for effectivelyimparting EDL-mediated effects to the inventive output materials.

In embodiments in which the inside surface 705 and the outside surface606 are constructed from a metallic material, such as stainless steel,the motion of the liquid 7120 and/or the streaming current(s) facilitateredox reactions involving H₂O, OH−, H+, and O₂ at the inside surface 705and the outside surface 606.

Referring to FIG. 27, without being limited by theory, it is believed asection 7140 of the mixing chamber 330 between the inside surface 705and the outside surface 606 the may be modeled as a pair of parallelplates 7142 and 7144. If the first material 110 is a liquid, the firstmaterial 110 enters the section 7140 through an inlet “IN” and exits thesection 7140 through an outlet “OUT.” The inlet “IN” and the outlet“OUT” restrict the flow into and out of the section 7140.

Referring to FIG. 28, the area between the parallel plates 7142 and 7144has a high surface area to volume ratio. Hence, a substantial portion ofthe counterion layer 7128 (and counterions 7126) may be in motion as thefirst material 110 moves between the plates 7142 and 7144. The number ofcounterions 7126 in motion may exceed the number allowed to enter thesection 7140 by the inlet “IN” and the number allowed to exit thesection 7140 by the outlet “OUT.” The inlet “IN” and the outlet “OUT”feeding and removing the first material 110 from the section 7140,respectively, have far less surface area (and a lower surface area tovolume ratio) than the parallel plates 7142 and 7144 and thereby reducethe portion of the counterions 7126 in motion in the first material 110entering and leaving the section 7140. Therefore, entry and exit fromthe section 7140 increases the streaming current locally. While abackground streaming current (identified by arrow “BSC”) caused by theflowing first material 110 over any surface is always present inside themixing device 100, the plates 7142 and 7144 introduce an increased“excess” streaming current (identified by arrow “ESC”) within thesection 7140.

Without a conductive return current (identified by arrow “RC”) in theplates 7142 and 7144 in the opposite direction of the flow of the firstmaterial 110, an excess charge 7146 having the same sign as theadsorbing ions 7122 would accumulate near the inlet “IN,” and an excesscharge 7148 having the same sign as the counterion 7126 would accumulatenear the at outlet “OUT.” Because such accumulated charges 7146 and7148, being opposite and therefore attracted to one another, cannotbuild up indefinitely the accumulated charges seek to join together byconductive means. If the plates 7142 and 7144 are perfectly electricallyinsulating, the accumulated charges 7146 and 7148 can relocate onlythrough the first material 110 itself. When the conductive returncurrent (identified by arrow “RC”) is substantially equivalent to theexcess streaming current (identified by arrow “ESC”) in the section7140, a steady-state is achieved having zero net excess streamingcurrent, and an electrostatic potential difference between the excesscharge 7146 near the inlet “IN,” and the excess charge 7148 near theoutlet “OUT” creating a steady-state charge separation therebetween.

The amount of charge separation, and hence the electrostatic potentialdifference between the excess charge 7146 near the inlet “IN,” and theexcess charge 7148 near the outlet “OUT,” depends on additional energyper unit charge supplied by a pump (e.g., the rotor 600, the internalpump 410, and/or the external pump 210) to “push” charge against theopposing electric field (created by the charge separation) to producethe a liquid flow rate approximating a flow rate obtainable by a liquidwithout ions (i.e., ions 7122 and 7126). If the plates 7142 and 7144 areinsulators, the electrostatic potential difference is a direct measureof the EMF the pump (e.g., the rotor 600, the internal pump 410 and/orthe external pump 210) can generate. In this case, one could measure theelectrostatic potential difference using a voltmeter having a pair ofleads by placing one of the leads in the first material 110 near theinlet “IN,” and the other lead in the first material 110 near the outlet“OUT.”

With insulating plates 7142 and 7144, any return current is purely anion current (or flow of ions), in that the return current involves onlythe conduction of ions through the first material 110. If otherconductive mechanisms through more conductive pathways are presentbetween the excess charge 7146 near the inlet “IN,” and the excesscharge 7148 near the outlet “OUT,” the return current may use those moreconductive pathways. For example, conducting metal plates 7142 and 7144may provide more conductive pathways; however, these more conductivepathways transmit only an electron current and not the ion current.

As is appreciated by those of ordinary skill, to transfer the chargecarried by an ion to one or more electrons in the metal, and vise versa,one or more oxidation-reduction reactions must occur at the surface ofthe metal, producing reaction products. Assuming the first material 110is water (H₂O) and the second material 120 is oxygen (O₂), anon-limiting example of a redox reaction, which would inject negativecharge into the conducting plates 7142 and 7144 includes the followingknown half-cell reaction:O₂+H₂O→O₃+2H⁺+2e ⁻,Again, assuming the first material 110 is water (H₂O) and the secondmaterial 120 is oxygen (O₂), a non-limiting example of a redox reactionincludes the following known half-cell reaction, which would removenegative charge from the conducting plates 7142 and 7144 includes thefollowing known half-cell reaction:2H⁺ +e ⁻→H₂,

With conducting metal plates 7142 and 7144, most of the return currentis believed to be an electron current, because the conducting plates7142 and 7144 are more conductive than the first material 110 (providedthe redox reactions are fast enough not to be a limiting factor). Forthe conducting metal plates 7142 and 7144, a smaller charge separationaccumulates between the inlet “IN” and the outlet “OUT,” and a muchsmaller electrostatic potential exists therebetween. However, this doesnot mean that the EMF is smaller.

As described above, the EMF is related to the energy per unit charge thepump provides to facilitate the flow of the first material 110 againstthe opposing electric field created by the charge separation. Becausethe electrostatic potential is smaller, the pump may supply less energyper unit charge to cause the first material 110 to flow. However, theabove example redox reactions do not necessarily occur spontaneously,and thus may require a work input, which may be provided by the pump.Therefore, a portion of the EMF (that is not reflected in the smallerelectrostatic potential difference) may be used to provide the energynecessary to drive the redox reactions.

In other words, the same pressure differentials provided by the pump topush against the opposing electric field created by the chargeseparation for the insulating plates 7142 and 7144, may be used both to“push” the charge through the conducting plates 7142 and 7144 and drivethe redox reactions.

Referring to FIG. 29, an experimental setup for an experiment conductedby the inventors is provided. The experiment included a pair ofsubstantially identical spaced apart 500 ml standard Erlenmeyer flasks7150 and 7152, each containing a volume of deionized water 7153. Arubber stopper 7154 was inserted in the open end of each of the flasks7150 and 7152. The stopper 7154 included three pathways, one each for ahollow tube 7156, a positive electrode 7158, and a negative electrode7160. With respect to each of the flasks 7150 and 7152, each of thehollow tube 7156, the positive electrode 7158, and the negativeelectrode 7160 all extended from outside the flask, through the stopper7154, and into the deionized water 7153 inside the flask. The positiveelectrode 7158 and the negative electrode 7160 were constructed fromstainless steel. The hollow tubes 7156 in both of the flasks 7150 and7152 had an open end portion 7162 coupled to a common oxygen supply7164. The positive electrode 7158 and the negative electrode 7160inserted into the flask 7152 where coupled to a positive terminal and anegative terminal, respectively, of a DC power supply 7168. Exactly thesame sparger was used in each flask.

Oxygen flowed through the hollow tubes 7156 into both of the flasks 7150and 7152 at a flow rate (Feed) of about 1 SCFH to about 1.3 SCFH(combined flow rate). The voltage applied across the positive electrode7158 and the negative electrode 7160 inserted into the flask 7152 wasabout 2.55 volts. This value was chosen because it is believed to be anelectrochemical voltage value sufficient to affect all oxygen species.This voltage was applied continuously over three to four hours duringwhich oxygen from the supply 7164 was bubbled into the deionized water7153 in each of the flasks 7150 and 7152.

Testing of the deionized water 7153 in the flask 7150 with HRP andpyrogallol gave an HRP-mediated pyrogallol reaction activity, consistentwith the properties of fluids produced with the alternate rotor/statorembodiments described herein. The HRP optical density was about 20%higher relative to pressure-pot or fine-bubbled solutions of equivalentoxygen content. The results of this experiment indicate that mixinginside the mixing chamber 330 involves a redox reaction. According toparticular aspects, the inventive mixing chambers provide for outputmaterials comprising added electrons that are stabilized by eitheroxygen-rich water structure within the inventive output solutions, or bysome form of oxygen species present due to the electrical effects withinthe process.

Additionally, the deionized water 7153 in both of the flasks 7150 and7152 was tested for both ozone and hydrogen peroxide employing industrystandard colorimetric test ampoules with a sensitivity of 0.1 ppm forhydrogen peroxide and 0.6 ppm for ozone. There was no positiveindication of either species up to the detection limits of thoseampoules.

Dwell Time

Dwell time is an amount of time the first material 110, the secondmaterial 120, and optionally the third material 130 spend in the mixingchamber 330. The ratio of the length of the mixing chamber 330 to thediameter of the mixing chamber 330 may significantly affect dwell time.The greater the ratio, the longer the dwell time. As mentioned in theBackground Section, the rotor 12 of the prior art device 10 (see FIG. 1)had a diameter of about 7.500 inches and a length of about 6.000 inchesproviding a length to diameter ratio of about 0.8. In contrast, inparticular embodiments, the length of the mixing chamber 330 of themixing device 100 is about 5 inches and the diameter “D1” of the rotor600 is about 1.69 inches yielding a length to diameter ratio of about2.95.

Dwell time represents the amount of time that the first material 110,the second material 120, and optionally the third material 130 are ableto interact with the electrokinetic phenomena described herein. Theprior art device 10 is configured to produce about 60 gallons of theoutput material 102 per minute and the mixing device 100 is configuredto produce about 0.5 gallons of the output material 102 per minute, theprior art device 10 (see FIG. 1) had a fluid dwell time of about 0.05seconds, whereas embodiments of the mixing device 100 have asubstantially greater (about 7-times greater) dwell time of about 0.35seconds. This longer dwell time allows the first material 110, thesecond material 120, and optionally the third material 130 to interactwith each other and the surfaces 606 and 705 (see FIG. 7) inside themixing chamber 330 for about 7 times longer than was possible in theprior art device 10.

With reference to Table I below, the above dwell times were calculatedby first determining the flow rate for each device in gallons persecond. In the case of the prior art device 10 was configured to operateat about 60 gallons of output material per minute, while the mixingdevice 100 is configured to operate over a broader range of flow rate,including at an optimal range of about 0.5 gallons of output materialper minute. The flow rate was then converted to cubic inches per secondby multiplying the flow rate in gallons per second by the number ofcubic inches in a gallon (i.e., 231 cubic inches). Then, the volume(12.876 cubic inches) of the channel 32 of the prior art device 10 wasdivided by the flow rate of the device (231 cubic inches/second) toobtain the dwell time (in seconds) and the volume (0.673 cubic inches)of the mixing chamber 330 of the mixing device 100 was divided by theflow rate (1.925 cubic inches/second) of the device (in cubic inches persecond) to obtain the dwell time (in seconds).

TABLE I Table 1. Inventive device can accommodate a range of dwelltimes, including a substantially increased (e.g., 7-times) dwell timerelative to prior art devices. Volume Flow Rate Mixing Flow Rate FlowRate Cubic Chamber Dwell Gallons/ Gallons/ Inches/ (Cubic Time DeviceMinute Second Second Inches) (Seconds) Prior art 60 1.000 231.000 12.8760.056 device 10 Mixing 2 0.033 7.700 0.673 0.087 device 100 Mixing 0.50.008 1.925 0.673 0.350 device 100

Rate of Infusion

Particular aspects of the mixing device 100 provide an improved oxygeninfusion rate over the prior art, including over prior art device 10(see FIG. 1). When the first material 110 is water and the secondmaterial 120 is oxygen, both of which are processed by the mixing device100 in a single pass (i.e., the return block of FIG. 2 is set to “NO”)at or near 20° Celsius, the output material 102 has a dissolved oxygenlevel of about 43.8 parts per million. In certain aspects, an outputmaterial having about 43.8 ppm dissolved oxygen is created in about 350milliseconds via the inventive flow through the inventive nonpressurized (non-pressure pot) methods. In contrast, when the firstmaterial 110 (water) and the second material 120 (oxygen) are bothprocessed in a single pass at or near 20° Celsius by the prior artdevice 10, the output material had dissolved oxygen level of only 35parts per million in a single pass of 56 milliseconds.

Output Material 102

When the first material 110 is a liquid (e.g., freshwater, saline,GATORADE®, and the like) and the second material 120 is a gas (e.g.,oxygen, nitrogen, and the like), the mixing device 100 may diffuse thesecond material 120 into the first material 110.

The following discusses results of analyses performed on the outputmaterial 102 to characterize one or more properties of the outputmaterial 102 derived from having been processed by the mixing device100.

When the first material 110 is saline solution and the second material120 is oxygen gas, experiments have indicated that a vast majority ofoxygen bubbles produced within the saline solution are no greater than0.1 micron in size.

Decay of Dissolved Oxygen Levels

Referring now to FIG. 30, there is illustrated the DO levels in waterenriched with oxygen in the mixing device 100 and stored in a 500 mlthin-walled plastic bottle and a 1000 ml glass bottle out to at least365 days. Each of the bottles was capped and stored at 65 degreesFahrenheit. As can be seen in the Figure, the DO levels of theoxygen-enriched fluid remained fairly constant out to at least 365 days.

Referring to FIG. 31, there is illustrated the DO levels in waterenriched with oxygen in the mixing device 100 and stored in a 500 mlplastic thin-walled bottle and a 1000 ml glass bottle. Both bottles wererefrigerated at 39 degrees Fahrenheit. Again, DO levels of theoxygen-enriched fluid remained steady and decreased only slightly out toat least 365 days.

Referring now to FIG. 32, there is illustrated the dissolved oxygenlevels in GATORADE® enriched with oxygen in the mixing device 100 andstored in 32 oz. GATORADE® bottles having an average temperature of 55degrees Fahrenheit at capping. The GATORADE® bottles were subsequentlyrefrigerated at 38 degrees Fahrenheit between capping and opening.During the experiment, a different bottle was opened at 20, 60, and 90days, respectively, to measure the DO levels of the GATORADE® storedtherein.

The GATORADE® within a first group of GATORADE® bottles was processedwith oxygen in the mixing device 100 at approximately 56 degreesFahrenheit. The DO levels of the GATORADE® at bottling wereapproximately 50 ppm as indicated by point 8104. A first bottle wasopened at approximately 20 days, and the DO level of the GATORADE® wasdetermined to be approximately 47 ppm as indicated by point 8106. Asecond bottle was then opened at 60 days, and the DO level of theGATORADE® was measured to be approximately 44 ppm as indicated by point8108. Finally, a third bottle was opened at 90 days, and the DO level ofthe GATORADE® was determined to be slightly below 40 ppm as indicated bypoint 8110.

The GATORADE® within a second group of GATORADE® bottles was processedwith oxygen in the mixing device 100 at approximately 52 degreesFahrenheit. The initial DO level for GATORADE® stored in this group ofbottles was 45 ppm as illustrated by point 8112. The GATORADE® in thebottle opened at 20 days had a DO level of only slightly lower than 45ppm as indicated by point 8114. The second bottle of GATORADE® wasopened at 60 days and the GATORADE® therein had a DO level of slightlymore than 41 ppm. Finally, a third bottle of GATORADE® was opened at 90days and the GATORADE® therein had a DO level of approximately 39 ppm asshown by point 8116. As before, with respect to the water test in theplastic and glass bottles (see FIG. 31), it can be seen that the DOlevels remain at relatively high levels over the 90 day period andsubstantially higher than those levels present in normal (unprocessed)GATORADE® stored in 32 oz. GATORADE® bottles. Point 8010 is the levelcorresponding to inventive output fluid in a covered PET bottle.

FIG. 33 illustrates the DO retention of 500 ml of braun balanced saltsolution processed with oxygen in the mixing device 100 and kept atstandard temperature and pressure in an amber glass bottle. The DO levelof the solution before processing is 5 ppm. After processing in themixing device 100, the DO level was increased to approximately 41 ppm(illustrated as point 8202). An hour after processing, the DO leveldropped to approximately 40 ppm as indicated by point 8204. Two hoursafter processing, the DO level dropped to approximately 36 ppm asindicated by point 8206. The DO level dropped to approximately 34 ppmthree hours after processing as indicated by point 8208. Atapproximately four and a half hours after processing, the DO levelwithin the salt solution dropped to slightly more than 30 ppm. The finalmeasurement was taken shortly before six hours after processing whereatthe DO level had dropped to approximately 28 ppm. Thus, each of theexperiments illustrated in FIGS. 30-33 illustrate that that the DOlevels remain at relatively high levels over extended periods.

Because the output material 102 may be consumed by human beings, thematerials used to construct the mixing device 100 should be suitable forfood and/or pharmaceutical manufacture. By way of non-limiting example,the housing 520, the housing 5520, the rotor 600, the stator 700, andthe stator 5700 may all be constructed from stainless steel.

Bubble Size Measurements

Experimentation was performed to determine a size of the bubbles of gasdiffused within the fluid by the mixing device 100. While experimentswere not performed to measure directly the size of the bubbles,experiments were performed that established that the bubble size of themajority of the gas bubbles within the fluid was smaller than 0.1microns. In other words, the experiments determined a size thresholdvalue below which the sizes of the majority of bubbles fall.

This size threshold value or size limit was established by passing theoutput material 102 formed by processing a fluid and a gas in the mixingdevice 100 through a 0.22 filter and a 0.1 micron filter. In performingthese tests, a volume of the first material 110, in this case, a fluid,and a volume of the second material 120, in this case, a gas, werepassed through the mixing device 100 to generate a volume of the outputmaterial 102 (i.e., a fluid having a gas diffused therein). Sixtymilliliters of the output material 102 was drained into a 60 ml syringe.The DO level of the fluid was measured via the Winkler Titration. Thefluid within the syringe was injected through a 0.22 micron filter intoa 50 ml beaker. The filter comprised the Milipor Millex GP50 filter. TheDO level of the material in the 50 ml beaker was then measured. Theexperiment was performed three times to achieve the results illustratedin Table II below.

TABLE II DO AFTER 0.22 DO IN SYRINGE MICRON FILTER 42.1 ppm 39.7 ppm43.4 ppm 42.0 ppm 43.5 ppm 39.5 ppm

As can be seen, the DO levels measured within the syringe and the DOlevels measured within the 50 ml beaker were not changed drastically bypassing the output material 102 through the 0.22 micron filter. Theimplication of this experiment is that the bubbles of dissolved gaswithin the output material 102 are not larger than 0.22 micronsotherwise there would be a significantly greater reduction in the DOlevels in the output material 102 passed through the 0.22 micron filter.

A second test was performed in which the 0.1 micron filter wassubstituted for the 0.22 micron filter. In this experiment, salinesolution was processed with oxygen in the mixing device 100 and a sampleof the output material 102 was collected in an unfiltered state. The DOlevel of the unfiltered sample was 44.7 ppm. The output material 102 wasfiltered using the 0.1 micron filter and two additional samples werecollected. The DO level of the first sample was 43.4 ppm. The DO levelof the second sample was 41.4 ppm. Then, the filter was removed and afinal sample was taken from the unfiltered output material 102. Thefinal sample had a DO level of 45.4 ppm. These results were consistentwith those seen using the Millipore 0.22 micron filter. These resultslead to the conclusion that there is a trivial reduction in the DOlevels of the output material 102 passed through the 0.1 micron filterproviding an indication that the majority of the bubbles in theprocessed saline solution are no greater than 0.1 micron in size.

As appreciated in the art, the double-layer (interfacial) (DL) appearson the surface of an object when it is placed into a liquid. Thisobject, for example, might be that of a solid surface (e.g., rotor andstator surfaces), solid particles, gas bubbles, liquid droplets, orporous body. In the mixing device 100, bubble surfaces represent asignificant portion of the total surface area present within the mixingchamber that may be available for electrokinetic double-layer effects.Therefore, in addition to the surface area and retention time aspectsdiscussed elsewhere herein, the relatively small bubble sizes generatedwithin the mixer 100 compared to prior art devices 10, may alsocontribute, at least to some extent, to the overall electrokineticeffects and output fluid properties disclosed herein. Specifically, inpreferred embodiments, as illustrated by the mixer 100, all of the gasis being introduced via apertures on the rotor (no gas is beingintroduced through stator apertures. Because the rotor is rotating at ahigh rate (e.g., 3,400 rpm) generating substantial shear forces at andnear the rotor surface, the bubble size of bubbles introduced via, andadjacent to the spinning rotor surface apertures would be expected to besubstantially (e.g., 2 to 3-times smaller) smaller than those introducedvia and near the stationary stator. The average bubble size of the priorart device 10 may, therefore, be substantially larger because at leasthalf of the gas is introduced into the mixing chamber from thestationary stator apertures. Because the surface area of a spheresurface varies with r², any such bubble component of the electrokineticsurface area of the mixing device 100 may be substantially greater thanthat of the prior art diffusion device 10.

Therefore, without being bound by theory, not only does the mixingchamber of the mixing device 100 have (i) a substantially higher surfaceto volume ratio than that of the prior art device 10 (the prior artdevice 10 has a ratio of surface to volume of 10.9, whereas the presentmixer 100 has a surface to volume ratio of 39.4), along with (ii) a7-fold greater dwell-time, but (iii) the unique properties of thecurrent output solutions may additionally reflect a contribution fromthe substantially larger bubble surface area in the mixing device 100.These distinguishing aspects reflect distinguishing features of thepresent mixing device 100, and likely each contribute to the uniqueelectrokinetic properties of the inventive output materials/fluids.

Sparging Effects

FIGS. 34-35 illustrate the sparging affects of the mixing device 100 ona fluid (e.g., the first material 110) passing therethrough. Spargingrefers to “bubbling” an inert gas through a solution to remove adifferent dissolved gas(es) from the solution. In each of the examplesillustrated in FIGS. 34 and 35, the second material 120 is nitrogen. Thelevels of dissolved oxygen in the output material 102 are measured atvarious points in time. As can be seen in the figures, the nitrogen gassparges the oxygen from the fluid passing through the mixing device 100causing the DO levels in the fluid to decay over a period of time.

The results of another experiment are illustrated in FIG. 34 whereinwater is sparged with nitrogen using the mixing device 100. Two sets ofexperiments were conducted, the first having a gas flow rate of SCFH(Standard Cubic Feet per Hour) of 1 and the second having a gas flowrate of SCFH of 0.6 The fluid flow rate was about 0.5 gal/min. As can beseen, when the process is begun, the DO levels in each of theexperiments was approximately 9 ppm. After only one minute, the DOlevels had dropped to slightly above 5 ppm. At two minutes the DO levelshad dropped to approximately 2.5 ppm. The DO level appears to level outat a minimum level at approximately 6 minutes wherein the DO level isslightly above zero (0). Thus, the nitrogen sparges the oxygen from thewater relatively quickly.

FIG. 35 illustrates the sparging of oxygenated water in an 8 gallon tankat standard temperature and pressure. The decay rate of the DO in thewater is illustrated by line 8602. As can be seen, initially theoxygenated water had a DO level of approximately 42 ppm. After 2 minutesof processing by the mixing device 100, the nitrogen sparged theoxygenated water such that the DO level dropped to slightly more than 20ppm. At 6 minutes, the DO level dropped from greater than 40 ppm to only6 ppm. The DO level of the oxygenated water reached a minimum valueslightly greater than zero (0) at approximately 14 minutes after thebeginning of the process. Thus, the above described sparging experimentsillustrate that the mixing device 100 is capable of quickly spargingoxygen from water and replacing the oxygen with another gas such asnitrogen by processing oxygenated water with mixing device 100 for arather short period of time. In other words, because total partial gaspressure in the fluid remained at approximately the same level despitethe decrease in DO, the nitrogen gas replaced the oxygen in the fluid.

These figures illustrate the manner in which nitrogen may be diffusedinto water to sparge the oxygen from the water. However, any gas couldbe used to sparge a selected gas from any selected fluid and diffuseinto the selected fluid the gas used to sparge the selected gas from theselected fluid. For example, the principals illustrated may also beapplicable to sparging nitrogen from water or another fluid usingoxygen. Further, any gas dissolved within a solution may be spargedtherefrom using a different gas to take the place of the gas spargedfrom the solution. In other words, by processing a sparging gas and asolution containing a dissolved gas through the mixing device 100 for arelatively short period of time, the dissolved gas could be quickly andefficiently removed from the solution.

Molecular Interactions

A number of physicists have begun to describe the quantum properties ofwater. Conventionally, quantum properties are thought to belong toelementary particles of less than 10⁻¹⁰ meters, while the macroscopicworld of our everyday life is referred to as classical, in that itbehaves according to Newton's laws of motion. Between the macroscopicclassical world and the microscopic quantum world is the mesoscopicdomain, where the distinction between macroscopic and microscopic isbecoming increasingly blurred. Indeed, physicists are discoveringquantum properties in large collections of atoms and molecules in thenanometer to micrometer range, particularly when the molecules arepacked closely together in a liquid phase.

Recently, chemists have made a surprising discovery that molecules formclusters that increase in size with dilution. These clusters measureseveral micrometers in diameter. The increase in size occursnon-linearly with dilution and depends on history, flying in the face ofclassical chemistry. Indeed, there is yet no explanation for thisphenomena. It may well be yet another reflection of the strangeness ofwater that depends on its quantum properties.

In the mid 1990's, quantum physicist del Giudice and Preparata and othercolleagues at the University of Milan, in Italy, argued that quantumcoherent domains measuring 100 nanometers in diameter could arise inpure water. They show how the collective vibrations of water moleculesin the coherent domain eventually become phase locked to thefluctuations of the global electromagnetic field. In this way, longlasting, stable oscillations could be maintained in water.

One way in which memory might be stored in water is through theexcitation of long lasting coherent oscillations specific to one or moresubstances (such as a therapeutic agent) dissolved in the water.Interactions between the water molecules and the molecules of thesubstances dissolved in the water change the collective structure of thewater, which would in turn determine the specific coherent oscillationsthat develop. If these oscillations become stabilized and maintained byphase coupling between the global field and the excited molecules, then,even when the dissolved substances are diluted away, the water may stillcarry the coherent oscillations that can seed other volumes of water ondilution.

The discovery that dissolved substances form increasingly large clustersis compatible with the existence of a coherent field in water that cantransmit attractive resonance between molecules when the oscillationsare in phase leading to clumping in dilute solutions. As a cluster ofmolecules increases in size, its electromagnetic signature iscorrespondingly amplified, reinforcing the coherent oscillations carriedby the water.

One should expect changes in some physical properties in water thatcould be detectable. Unfortunately, all attempts to detect such coherentoscillations by usual spectroscopic and nuclear magnetic resonancemethods have yielded ambiguous results. This is not surprising in viewof the finding that cluster size of the dissolved molecules depends onthe precise history of dilution rather than concentration of themolecules.

It is possible that despite variations in the cluster size of thedissolved molecules and detailed microscopic structure of the water, aspecificity of coherent oscillations may nonetheless exist. Usualdetection methods fail because they depend upon using the microscopicparticles of individual molecules, or of small aggregates. Instead, whatis needed is a method of detecting collective global properties overmany, many molecules. Some obvious possibilities that suggest themselvesare the measurements of freezing points and boiling points, viscosity,density, diffusivity, and magnet properties. One possibility fordetecting changes in collective global properties of water is by meansof crystallization. Crystals are formed from macroscopic collections ofmolecules. Like other measurements that depend on global properties,crystals simplify the subtle changes in the individual molecules thatwould have been undetectable otherwise.

With reference to FIG. 36, a simplified protonated water cluster forminga nanoscale cage 8700 is shown. A protonated water cluster typicallytakes the form of H⁺(H₂O)_(n). Some protonated water clusters occurnaturally, such as in the ionosphere. Without being bound by anyparticular theory, and according to particular aspects, other types ofwater clusters or structures (clusters, nanocages, etc) are possible,including structures comprising oxygen and stabilized electrons impartedto the inventive output materials. Oxygen atoms 8704 may be caught inthe resulting structures 8700. The chemistry of the semi-bound nanocageallows the oxygen 8704 and/or stabilized electrons to remain dissolvedfor extended periods of time. Other atoms or molecules, such asmedicinal compounds, can be caged for sustained delivery purposes. Thespecific chemistry of the solution material and dissolved compoundsdepend on the interactions of those materials.

Fluids processed by the mixing device 100 have been shown viaexperiments to exhibit different structural characteristics that areconsistent with an analysis of the fluid in the context of a clusterstructure.

Rayleigh Effects

If a strong beam of light is passed through a transparent gaseous orliquid medium containing solid or liquid particles, or even molecules ofextremely high molecular weight, the light is scattered away from thedirection of its incident path. The scattering is due to theinterference effects that arise from the density fluctuations in thescattering medium (i.e. the presence of particles or very high molecularweight molecules.) There are two types of light scattering. The firstinvolves the wavelength of the scattered light differing from that ofthe incident light and is called Raman scattering. The other typescattering involves when the scattered light has the same wavelength ofthe incident light and is called Rayleigh scattering. In Rayleighscattering, the intensity of the scattered light is proportional to theproduct of the intensity of the incident light and the attenuationconstant, a function of the refractive index and the Rayleigh constant.The Rayleigh constant is a somewhat involved function of the molecularweight of the scattering substance and thus a measurement of theintensity of the scattered light can give a value for the molecularweight. This scattering phenomenon is used in a number of liquidchromatography detectors.

Water processed through the mixing device 100 has been demonstrated tohave detectable structural differences when compared with normalunprocessed water. For example, processed water has been shown to havemore Rayleigh scattering than is observed in unprocessed water. In theexperiments that were conducted, samples of processed and unprocessedwater were prepared (by sealing each in a separate bottle), coded (forlater identification of the processed sample and unprocessed sample),and sent to an independent testing laboratory for analysis. Only afterthe tests were completed were the codes interpreted to reveal whichsample had been processed by the mixing device 100.

At the laboratory, the two samples were placed in a laser beam having awavelength of 633 nanometers. The fluid had been sealed in glass bottlesfor approximately one week before testing. With respect to the processedsample, Sample B scattered light regardless of its position relative tothe laser source. However, “Sample A” did not. After two to three hoursfollowing the opening of the bottle, the scattering effect of Sample Bdisappeared. These results imply the water exhibited a memory causingthe water to retain its properties and dissipate over time. Theseresults also imply the structure of the processed water is opticallydifferent from the structure of the unprocessed fluid. Finally, theseresults imply the optical effect is not directly related to DO levelsbecause the DO level at the start was 45 ppm and at the end of theexperiment was estimated to be approximately 32 ppm.

Generation of Solvated Electrons

Additional evidence indicates that the mixing occurring inside themixing device 100 generates solvated electrons within the outputmaterial 102. This conclusion results from conditions observed withrespect to the dissolved oxygen probe effects used in measuring the DOlevels within various processed solutions. Due to the experiences viewedwith respect to the polarographic dissolved oxygen probes, it is abelief that the processed fluid exhibits an electron capture effect andthus the fluid includes solvated electrons.

There are two fundamental techniques for measuring dissolved oxygen(“DO”) levels electrically: galvanic measuring techniques andpolarographic measurements. In both techniques, the DO level sensorincludes two electrodes, an anode and a cathode, which are both immersedin electrolyte within the sensor body. An oxygen permeable membraneseparates the anode and cathode from the solution being tested. Thecathode is a hydrogen electrode and carries negative potential withrespect to the anode. The electrolyte solution surrounds the electrodepair and is contained by the membrane. With no oxygen, the cathodebecomes polarized with hydrogen and resists the flow of current. Whenoxygen passes through the membrane, the cathode is depolarized andelectrons are consumed. In other words, oxygen diffuses across themembrane and interacts with the internal components of the probe toproduce an electrical current. The cathode electrochemically reduces theoxygen to hydroxyl ions according to the following equation:O₂+2H₂O+4E ⁻=4OH⁻

When attempting to measure DO levels in a solution processed by themixing device 100, an overflow condition has been repeatedly experiencedwherein the dissolved oxygen meter actually displays a reading that ishigher than the meter is capable of reading. Independent means, aWinkler Titration, reveals a much lower DO level for the solution thanindicated by the probe. Typically, in a device such as the Orion 862,having a maximum reading of 60 ppm, the meter will overflow and have thehigh oxygen level indication if left in bulk processed water for severalminutes. Because the overload is not caused by dissolved oxygen in thefluid, it is believed solvated electrons must be causing the overload.In other words, solvated electrons are accompanying the processed wateracross the membrane. These electrons are attracted to the anode andcause the current observed. It is a further belief that these electronsare captured in a cage or cluster mechanism within the solution.

Compositions Comprising Hydrated (Solvated) Electrons Imparted to theInventive Compositions by the Inventive Processes

In certain embodiments as described herein (see under “Double-layer”),the gas-enriched fluid is generated by the disclosed electromechanicalprocesses in which molecular oxygen is diffused into the fluid and mayoperate to stabilize charges (e.g., hydrated (solvated) electrons)imparted to the fluid. Without being bound by theory or mechanism,certain embodiments of the present invention relate to a oxygen-enrichedfluid (output material) comprising charges (e.g., hydrated (solvated)electrons) that are added to the materials as the first material ismixed with oxygen in the inventive mixer device to provide the combinedoutput material. According to particular aspects, these hydrated(solvated) electrons (alternately referred to herein as ‘solvatedelectrons’) are stabilized in the inventive solutions as evidenced bythe persistence of assayable effects mediated by these hydrated(solvated) electrons. Certain embodiments may relate to hydrated(solvated) electrons and/or water-electron structures, clusters, etc.,(See, for example, Lee and Lee, Bull. Kor. Chem. Soc. 2003, v. 24, 6;802-804; 2003).

Novel HRP based assay. Horseradish peroxidase (HRP) is isolated fromhorseradish roots (Amoracia rusticana) and belongs to theferroprotoporphyrin group (Heme group) of peroxidases. HRP readilycombines with hydrogen peroxide or other hydrogen donors to oxidize thepyrogallol substrate. Additionally, as recognized in the art, HRPfacilitates autoxidative degradation of indole-3-acietic acid in theabsence of hydrogen peroxide (see, e.g., Heme Peroxidases, H. BrianDunford, Wiley-VCH, 1999, Chapter 6, pages 112-123, describing thatautoxidation involves a highly efficient branched-chain mechanism;incorporated herein by reference in its entirety). The HRP reaction canbe measured in enzymatic activity units, in which Specific activity isexpressed in terms of pyrogallol units. One pyrogallol unit will form1.0 mg purpurogallin from pyrogallol in 20 sec at pH 6.0 at 20° C. Thispurpurogallin (20 sec) unit is equivalent to approx. 18 μM units per minat 25° C.

According to particular aspects of the present invention, theoxygen-enriched inventive fluids (output materials) have been describedand disclosed herein to react with pyrogallol in the presence ofhorseradish peroxidase. The reaction is most likely based on anauto-oxidation of the pyrogallol, since no hydrogen peroxide,superoxide, or other reactive oxygen species has been detected inoxygen-enriched inventive fluid. The extent of this reaction is greaterthan that of pressurized oxygen solutions (pressure-pot oxygensolutions) and less than that of hydrogen peroxide.

Specifically, the present applicants have determined that while there isno hydrogen peroxide (none detected at a sensitivity of 0.1 ppm), theinventive gas-enriched fluid may be consistently characterized by itsfacilitation of the apparent autoxidation of pyrogallol to purpurogallinin the presence of horseradish peroxidase enzyme (HRP). That is, likethe case of HRP facilitation of the autoxidative degradation ofindole-3-acietic acid in the absence of hydrogen peroxide, applicantshave discovered HRP facilitation of the autoxidative degradation ofpyrogallol in the absence of hydrogen peroxide. According to particularaspects, the presence and level of this activity are distinguishingfeatures of the inventive compositions in view of the prior art.

In certain embodiments, the inventive gas-enriched fluid facilitates, inthe presence of HRP and absence of hydrogen peroxide, a pyrogallolautoxidation rate (under standard conditions as defined herein under“Definitions”) equivalent to approximately 0.5 ppm of hydrogen peroxide,approximately 0.8 ppm of hydrogen peroxide, approximately 1 ppm ofhydrogen peroxide, approximately 2 ppm of hydrogen peroxide,approximately 3 ppm of hydrogen peroxide, approximately 4 ppm ofhydrogen peroxide, approximately 5 ppm of hydrogen peroxide,approximately 6 ppm of hydrogen peroxide, approximately 7 ppm ofhydrogen peroxide, approximately 8 ppm of hydrogen peroxide,approximately 9 ppm of hydrogen peroxide, approximately 10 ppm ofhydrogen peroxide, approximately 11 ppm of hydrogen peroxide,approximately 12 ppm of hydrogen peroxide, approximately 20 ppm ofhydrogen peroxide, approximately 40 ppm of hydrogen peroxide,approximately 50 ppm of hydrogen peroxide or any value therebetween orgreater.

It is known that Horseradish peroxidase enzyme catalyzes theauto-oxidation of pyrogallol by way of facilitating reaction with themolecular oxygen in a fluid. (Khajehpour et al., PROTEINS: Struct,Funct, Genet. 53: 656-666 (2003)). It is also known that oxygen bindsthe heme pocket of horseradish peroxidase enzyme through a hydrophobicpore region of the enzyme (between Phe68 and Phe142), whose conformationlikely determines the accessibility of oxygen to the interior. Withoutbeing bound by mechanism, because surface charges on proteins are knownin the protein art to influence protein structure, it is possible thatthe solvated electrons present in the inventive gas-enriched fluid actto alter the conformation of the horseradish peroxidase such thatgreater oxygen accessibility results. The greater accessibility ofoxygen to the prosthetic heme pocket of the horseradish peroxidaseenzyme in turn would allow for increased reactivity with pyrogallol,when compared with prior art oxygenated fluids (pressure-pot,fine-bubbled). Alternatively, the added or solvated electrons of thepresent output compositions may be acting in other ways to enablefacilitation of the apparent autoxidation of pyrogallol to purpurogallinin the presence of horseradish peroxidase enzyme (HRP).

In any event, according to particular aspects, production of outputmaterial using the inventive methods and devices comprises a processinvolving: an interfacial double layer that provides a charge gradient;movement of the materials relative to surfaces pulling charge (e.g.,electrons) away from the surface by virtue of a triboelectric effect,wherein the flow of material produces a flow of solvated electrons.Moreover, according to additional aspects, and without being bound bymechanism, the orbital structure of diatomic oxygen creates chargeimbalances (e.g., the two unpaired electrons affecting the hydrogenbonding of the water) in the hydrogen bonding arrangement within thefluid material (water), wherein electrons are solvated and stabilizedwithin the imbalances.

The inventive combination of oxygen-enrichment and solvated electronsimparted by the double-layer effects and configuration of the presentlyclaimed devices facilitates the auto-oxidation of pyrogallol in thepresence of HRP, which is a distinguishing feature of the presentinventive output material compositions that can be readily monitored andquantified by way of optical density. Typically, the inventiveoxygen-enriched compositions are characterized in that they provide forabout a 20% higher optical density read-out in the standard assaycompared to either pressurized (pressure pot) or fine-bubbled controlfluid have equivalent dissolved oxygen concentrations. The HRP is likelyproviding added oxidative ability to the autoxidation.

Pyrogallol Reactivity Test

An aliquot of the inventive oxygen-enriched output material was testedfor peroxidase activity by using a commercially available horseradishperoxidase and a pyrogallol assay (Sigma). Briefly, pyrogallol stocksolution was prepared with deionized water. Pyrogallol measuresperoxidase activity of the horseradish peroxidase enzyme on the fluid asit reacts with a substrate (such as hydrogen peroxide), to yieldpurpurogallin and water. Test fluid with horseradish peroxidase,pyrogallol and the appropriate potassium phosphate buffer were comparedwith other fluids. Hydrogen peroxide served as the positive control. Theother fluids tested were water that was oxygenated and pressurized in apressure pot, up to 100 psi to reach the desired dissolved oxygen level(Pressure Pot), while the other fluid was oxygenated with an air stonein an open beaker (Fine Bubble). All fluids tested were maintained atroom temperature, and measured approximately 55 ppm dissolved oxygenlevel (by FOXY probe). Water samples were tested by adding the enzymaticreagents. Continuous spectrophotometric rate determination was made atA₄₂₀ nm, and room temperature (25 degrees Celsius).

As indicated in FIGS. 38-41, the inventive oxygen-enriched fluid testedpositive for reactivity with horseradish peroxidase by pyrogallol, whilethe pressure pot and fine bubbled water samples had far less reactivity.As indicated in FIG. 42, oxygen is required for the reaction withpyrogallol in the presence of horseradish peroxidase, as inventive fluidenriched with other gases did not react in the same manner.

Several chemical tests of the inventive oxygen-enriched fluid for thepresence of hydrogen peroxide were conducted, as described herein, andnone of these tests were positive (sensitivity of 0.1 ppm hydrogenperoxide). Thus, the inventive oxygen-enriched fluid of the instantapplication provides for peroxidase facilitated auto-oxidation activityin the absence of hydrogen peroxide.

In particular embodiments, Applicants have determined that thehorseradish peroxidase effect remains at least up to seven hours afteropening of the bottle in which it is stored. In other embodiments,Applicants have determined that the horseradish peroxidase effectremains after opening of closed container after 105 days of storage inthe closed container. By contrast, in other embodiments, Applicants havedetermined that when testing equivalent dissolved oxygen levels madewith just pressurizing fluid (pressure pot fluids), the decline of abackground HRP effect takes place rapidly, declining precipitously inunder 4 hours.

Glutathione Peroxidase Study

The inventive oxygen-enriched output fluid material was tested for thepresence of hydrogen peroxide by testing the reactivity with glutathioneperoxidase using a standard assay (Sigma). Briefly, glutathioneperoxidase enzyme cocktail was constituted in deionized water and theappropriate buffers. Water samples were tested by adding the enzymaticreagents. Continuous spectrophotometric rate determination was made atA₃₄₀ nm, and room temperature (25 degrees Celsius). Samples testedwere: 1. deionized water (negative control), 2. inventiveoxygen-enriched fluid at low concentration, 3. inventive oxygen-enrichedfluid at high concentration, 4. hydrogen peroxide (positive control). Asillustrated in FIG. 43, the hydrogen peroxide positive control showed astrong reactivity, while none of the other fluids tested reacted withthe glutathione peroxidase.

Differential Nucleic Acid Stability

Particular embodiments of the present invention provide anotherdistinguishing feature of the present inventive compositions.Specifically, applicants have discovered that there is a differentialthermostability of nucleic acids associated with the inventive outputfluids compared to control fluids. For example, the T7 promoter primer5′-d(TAATACGACTCACTATAGGG)-3′ (SEQ ID NO:1) when measured in theinventive oxygen-enriched output materials relative to non-enricheddeionized water. As the temperature of the water increases, the DNAoligomeric structure performs a conformational change. As illustrated inFIG. 44, consent with the art recognized melting temperature for thisoligo of about 48° C., the T7 DNA begins to denature at about 50 degreesCelsius in the control (deionized water), whereas the DNA in theoxygen-enriched inventive fluid remains intact until about 60 degreesCelsius. Thus, the inventive oxygen-enriched fluid comprising solvatedelectrons imparts a higher thermostability for DNA when compared tocontrol fluid, and provides a further distinguishing feature of thepresent inventive output material compositions that can be readilymonitored and quantified by way of optical density measurements.

Bioreactor Systems Comprising the Inventive Mixing Devices

Producing significant quantities of target products, such as proteins,polypeptides, nucleic acids, therapeutic agents, and other products inhost cell systems are possible due to advances in molecular biology. Forexample, recombinant proteins are produced in a host cell systems bytransfecting the host cell with nucleic acids (e.g. DNA) encoding aprotein of interest. Next, the host cell is grown under conditions whichallow for expression of the recombinant protein. Certain host cellsystems can be used to produce large quantities of recombinant proteinswhich would be too impractical to produce by other means.

In addition, enzymatic and/or reaction fermentations, with or withouthost cells, are utilized for example in producing foodstuff andbeverages, in treating wastewater, or in environmental cleanup.

Cell culturing processes, or cellular fermentation, typically useprokaryotic or eukaryotic host cells to produce recombinant proteins.The fermentation is typically conducted in physical containers (e.g.stirred tanks) called fermentors or tank bioreactors. The fermentationprocess itself may comprise (1) discontinuous operation (batch process),(2) continuous operation, or (3) semi-continuous operations (such as thefed-batch process), or any combination of these.

Since the aim of large scale production of pharmaceutical drugs (e.g.biologicals) or other target products is to provide improvedmanufacturing processes and reduced costs, there is a need for improvedbioreactor equipment, methods, and media for preparation of these targetproducts.

The present disclosure sets forth novel gas-enriched fluids, including,but not limited to gas-enriched water, saline solutions (e.g., standardaqueous saline solutions), cell culture media, as well as novel methodsand biological and chemical reactor systems for use in these applicationprocesses, and others.

Certain embodiments disclosed herein relate to systems, media, andmethods for producing a target product, such as a protein.

In certain embodiments, a target product may refer to a protein,peptide, polypeptide, nucleic acid, carbohydrate, polymer, micelle, andany mixture thereof.

The target product is typically produced by a vehicle, such as a hostcell, which is associated with the gas-enriched fluid in a chemical orbiological reactor. Reactors may include standard reactors, such ascontinuous feed, discontinuous feed, and/or semi-continuous feed.Reactors may also include a cell culture vessel (such as a plate, flask,or tank), a plant, an animal, a fungus, an algae, or other organism. Forexample, a plant that is associated with the gas-enriched fluid of thepresent invention may comprise plant cells acting as vehicles that aidin the production of the target product (for example, naturallyoccurring plant matter or genetically altered plant matter).

In certain embodiments, the vehicles utilized with the gas-enrichedfluids or solutions (including media) may include prokaryotic cells oreukaryotic cells. More specifically, the living cells may includebacterial (e.g. E. coli, Salmonella, Streptococcus, Staphylococcus,Neisseria, Nocardia, Mycoplasma, etc.), fungal (e.g. yeasts, molds,mushrooms, etc.), plant (tobacco, maize, soybean, fruit or vegetable,etc.), animal (mammalian, insect, etc.) archebacterial (blue greenalgae), protist, human embryonic kidney (HEK) cells, HeLa cells,hybridoma cells, Madin-Darby Canine Kidney (MDCK) cells, stem cells,cell lines (including SP2/0 and NSO), African Green Monkey Kidney (Vero)cells, Spodoptera frugiperda (army worm), Trichoplusia ni (cabbagelooper), and other cells. In addition, viruses (such as bacteriophage,baculovirus, vaccinia, and other viruses) may be employed in thebioreactors of the present invention.

The bioreactor may comprise an airlift reactor, a packed bed reactor, afibrous bed reactor, a membrane reactor, a two-chamber reactor, astirred-tank reactor, a hollow-fiber reactor, or other reactor designedto support suspended or immobilized cell growth.

In cases of recombinant or target protein production, a balanced batchand/or feed medium must encourage optimal cell growth and expression ofthe recombinant protein. The medium, or media, is termed “minimal” if itonly contains the nutrients essential for growth. For prokaryotic hostcells, the minimal media typically includes a source of carbon,nitrogen, phosphorus, magnesium, and trace amounts of iron and calcium.(Gunsalus and Stanter, The Bacteria, V. 1, Ch. 1 Acad. Press Inc., N.Y.(1960)). Most minimal media use glucose as a carbon source, ammonia as anitrogen source, and orthophosphate (e.g. PO₄) as the phosphorus source.The media components can be varied or supplemented according to thespecific prokaryotic organism(s) grown, in order to encourage optimalgrowth without inhibiting target protein production. (Thompson et al.,Biotech. and Bioeng. 27: 818-824 (1985)). This allows for higher levelsof production with lower cost.

In addition to the chemical composition of the media, other factors mayaffect cell growth and/or target protein production. These factorsinclude, but are not limited to pH, time, cultivation temperature,amount of dissolved oxygen or other gas(es), and partial pressure ofthose dissolved gasses. During the fermentation process, the pH of themedia is typically altered due to the consumption of ammonia, ormicroorganism synthesis of certain metabolic products, e.g., acetic acidand lactic acid. Since altered pH may be unfavorable for optimal cellgrowth, it may be necessary or desirable to maintain the medium at acertain pH (i.e. by addition of acids or bases). The pH and otherprocess parameters can be monitored manually or by automatic devices.

Inventive Gas-Enriched Fluids

Enriching a fluid with another fluid may result in a solution orsuspension of the two fluids, depending on the physical and chemicalproperties of the two fluids. In particular, enriching a liquid with agas (e.g. oxygen) may be beneficial for certain applications, includingtherapeutic treatments. As utilized herein, “fluid,” may generally referto a liquid, a gas, a vapor, a mixture of liquids and/or gases, a liquidand/or gas solution, or any combination thereof, for any particulardisclosed embodiment. Furthermore, in certain embodiments a “liquid” maygenerally refer to a pure liquid or may refer to a gel, sol, emulsion,fluid, colloid, dispersion, suspension, or mixture, as well as anycombination thereof; any of which may vary in viscosity.

In particular embodiments, the dissolved gas comprises oxygen. In otherparticular embodiments, the dissolved gas comprises nitrogen, carbondioxide, carbon monoxide, ozone, sulfur gas, nitrous oxide, nitricoxide, argon, liquefied petroleum gas, helium, natural gas, or others.

One particular advantage of embodiments of the present invention relatesto the gas-enriched fluids' long-term diffused gas (particularly oxygen)levels, which allows for long-term bio-availability of the gas tocellular or chemical reactors. The long-term bio-availability of gassesin the gas-enriched fluids of the present invention allow for increasedtarget product production and/or improved enzymatic or other chemicalreactions that benefit from the gas-enriched fluids (includingoxygenated media) of the present invention.

In some instances, living cells may be grown in a bioreactor orfermentation chamber in order to promote cell growth and/or productionof the intended target product. While some living cells require amixture of gasses in order to sustain or promote their survival orpropagation, cell growth may be hindered or ceased if a particular gas,such as oxygen, is present at too high of a concentration.

For example, mammalian cells, such as Chinese Hamster Ovary (CHO) cells,require oxygen in order to proliferate. However, the existing techniquesin the art for diffusing gasses, such as oxygen, into the bioreactorfluids have a detrimental effect on mammalian cell cultures. Forexample, the cells may be destroyed or rendered non-viable in instanceswhere the diffused gas bubbles rupture or coalesce within the culturemedia, which is particularly common at a gas-to-liquid interface.Accordingly, the present invention represents an advance that would nothave occurred in the ordinary course since the existing knowledge in theart teaches that the levels of dissolved gas, particularly the levels ofdissolved oxygen, in the gas-enriched media disclosed herein ispredicted to be harmful or detrimental. However, the gas-enriched fluidmedia as described herein result in imparting at least one beneficialadvantage to cell cultures selected from the group consisting of:enhanced cell growth (e.g. rate and/or number) increased target productyield (e.g. amount), increased rate of target product production,improved vehicle cell viability, increased efficiency of target productproduction, increased ease in target product purification, and the like.In certain embodiments, one or more of these beneficial advantages areconveyed to cell cultures without proving injurious to the cellsthemselves.

In other embodiments, an acellular reaction may utilize the gas-enrichedfluids and methods of the present invention, including general chemicaland/or enzymatic reactions. Examples of such reactions include, but arenot limited to, wastewater treatment, purification of water (such astreating municipal water, home drinking purifiers, cleaning swimmingpools or aquariums, etc.), homogenization of milk, hydrogenation ofoils, gas-enriching fuels, and others.

In further embodiments, the gas-enriched fluid maintains a dissolved gasenrichment level of at least 10 ppm, at least 15 ppm, at least 20 ppm,at least 25 ppm, at least 30 ppm, at least 35 ppm, at least 40 ppm, atleast 45 ppm, at least 50 ppm, at least 55 ppm, at least 60 ppm, atleast 65 ppm, at least 70 ppm, at least 75 ppm, at least 80 ppm, atleast 85 ppm, at least 90 ppm, at least 100 ppm, or any value greater ortherebetween, at atmospheric pressure. In certain instances, thegas-enriched fluid maintains its dissolved gas enrichment level (i.e.the level of the gas enriched in the fluid) for a period of at least 10days, at least 20 days, at least 30 days, at least 40 days, at least 50days, at least 60 days, at least 70 days, at least 80 days, at least 90days, at least 100 days, at least 110 days, at least 120 days, at least130 days, or greater or any value therebetween, within a sealedcontainer at atmospheric pressure.

In one particular embodiment, the host material comprises water or watervapor. In another particular embodiment, the host material comprisesother fluids (i.e. gasses or liquids) such as wastewater, toxicmaterials, potable water, milk, juice, yogurt, soft drinks (particularlycarbonated beverages), ethanol, methanol, polymers (such as plastic orrubber compounds), oil (edible or non-edible), emulsions, suspensions,aqueous carriers, non-aqueous carriers, and the like.

In certain embodiments, multiple gasses may be used to enrich or infusea host fluid. In certain embodiments, ozone and/or oxygen may be used tobreak down complex structures into smaller substructures, particularlyif used with sonochemistry techniques, as described herein inter alia.

In certain embodiments, the gas-enriched fluid or other host material ofthe present invention has characteristics that may be more similar tothe gas that has enriched the fluid or other host material, or it mayhave characteristics that are more similar to the fluid (e.g. gas orliquid) or other host material itself.

In certain embodiments, a gas-enriched fluid or solution comprisesgas-enriched culture media. In particular embodiments, the gas-enrichedmedia comprises oxygenated or oxygen-enriched media. In certainembodiments, the gas-enriched fluid or gas-enriched host material mayinclude further processing, such as by filtering, separating, modifyingor altering various constituents of the fluid or host material.

Packaged Gas-Enriched Fluids

Certain embodiments disclosed herein relate to gas-enriched fluids thathave high levels of dissolved or diffused gases (particularly oxygen)that may be produced by various methods, including those describedherein. In certain embodiments, the gas-enriched fluid may be producedin a biomass production facility and applied directly to a bioreactorsystem. Alternatively, the gas-enriched fluid may be packaged anddistributed for use at other locations. In the event that thegas-enriched fluid is packaged, such packaging may include a sterilepackage such as a glass or plastic container, flexible foil or plasticpouches, sealed boxes (particularly waxed boxes), and the like. In thecase of sealed packages, the gas-enriched fluid may maintain a highlevel of dissolved or diffused gas for several days, several weeks, orseveral months. In certain embodiments, the sealed container (i.e.enclosed with a cap, cover or other enclosure that is at leastsemi-impermeable to gas exchange) maintains the diffused nature of thefluid at least 2 weeks, at least 4 weeks, at least 2 months, at least 4months, at least 6 months, at least 8 months, at least 10 months, atleast 12 months, or any value greater or therebetween.

Gas-Enriched Fluids in Biological or Chemical Reactors

As illustrated in FIGS. 45A and 45B, a biological or chemical reactorsystem 3300 a may be used for conventional large-scale cell-culturing orchemical processing to achieve the production of the target product3318. The target product 3318 may include, but not be limited to,proteins, antibodies, synthetic peptides, active pharmaceutical agents,foodstuff or beverage products (such as wine; beer; soft drinks; fruitor vegetable juices); plant products (flowers, cotton, tobacco, wood,paper, wood or paper pulp, etc.); ethanol, methanol, paints, fruit orvegetables or fruit or vegetable products such as jellies, jams, sauces,pastes, and the like; cheese or cheese products; nuts or nut products(such as peanut butter, almond paste, etc.); meat or meat products,grain flours or products including bread, cereal, pasta, and the like;slurries or mixtures of any of these, processed polymers (includingplastics, and other polymers), petroleum products, and others.

In certain embodiments, in the case of using a vessel reactor, such as atank reactor, the target product resides within inclusion bodies,particularly when E. coli cells are utilized. The target product may beobtained by processing the inclusion bodies, for example by usinghigh-pressure homogenizers or other techniques.

In particular embodiments in which the reactor is a biological reactorsystem, the system 3306 includes a source 3308 of culture cells 3310 tobe cultured, a source 3302 of culture media 3304, a biological reactor3306, and a harvesting and purification system 3316, for producing thetarget product 3318. The culture cells 3310 are geneticallypredetermined to produce proteins or the like that constitute the targetproduct 3318, and the culture medium 3304 may comprise a sterile mediumof a type that provides nourishment for the proliferation of culturecells 3310. In this particular exemplary embodiment, the sterile medium3304 is introduced into the internal chamber (which may be referred toas the “fermentation chamber”) of the reactor 3306 from the source 3302.From the source 3308, the culture cells 3310 are provided such that thecells 3310 and medium 3304 are combined into a broth 3312 in thefermentation chamber of the bioreactor 3306.

The appropriate base medium 3304 to be utilized in the reactor system3300 a may be formulated to provide optimal nourishment and growth tothe cell culture 3310. Medium 3304 is preferably a fluid (e.g. liquid orgas) medium, more preferably a liquid medium or a solid-liquid mediumthat is selected based on the certain variables, such as thecharacteristics and objectives of the overall bioreactor system 3300 a,the cost, the type of cells being cultured from the cell culture 3310,the desired production parameters, the type of culturing and mediamanagement process used in the reactor 3306, the type of downstreamharvest and purification processes 3316, and the target activepharmaceutical ingredient 3318. Various cell culture media presentlyused may be adapted for use or gas-enrichment by the present invention.

In certain embodiments, a suitable base medium 3304 may include but notbe limited to a serum-supplemented medium, a hydrolysate medium,chemically-synthesized medium, chemically-defined medium, a serum-freemedium, any combination of these or other media.

In certain embodiments, the gas-enriched media may be supplemented withtransferrins, albumins, fetuins, protein hydrolysates, or otheradditives, preservatives, nutrients, fillers, shear protectants (such asPluronic F68), or active or inactive agents.

In addition, the medium may be formulated for cells that are attached tosome type of support within the bioreactor 3306, rather than suspendedin the broth 3312. In all embodiments that utilize a medium 3304, themedium 3304 is formulated to meet the nutritional requirements of theindividual cell type in the cell culture 3310, and typically compriseminerals, salts, and sugars.

In certain embodiments, medium 3304 and/or broth 3312 are gas-enrichedusing the presently disclosed mixing devices 100, in order to dissolveor diffuse gases (such as oxygen) into, for example, the media, both orcomponents thereof, in concentrations of at least about 8 ppm, at leastabout 10 ppm, at least about 20 ppm, at least about 25 ppm, at leastabout 30 ppm, at least about 35 ppm, at least about 40 ppm, at leastabout 50 ppm, at least about 60 ppm, at least about 70 ppm, at leastabout 80 ppm, at least about 90 ppm, at least about 100 ppm, or anyvalue greater or therebetween. In certain embodiments, the gas-enrichedmedium and/or broth contains less than about 160 ppm.

In certain embodiments, the typical biological or chemical reactor isloaded with sterilized raw materials (nutrients, reactants, etc.) alongwith air or specific gas, as well as cells for a biological reactor.Other agents may be added to the mixture, including anti-foamingchemicals or agents, pH controlling agents, and/or other agents. Thetarget product is typically recovered by separating the cells, and/ordisrupting the cells in order to extract the product, concentrating theproduct, and purifying, drying, or further processing the product.

Many different types of bioreactor systems are in use today, any ofwhich can be used with the gas-enriched media of the present invention.For example, air-lift bioreactors are commonly used with bacteria, yeastand other fungi; fluidized-bed bioreactors are commonly used withimmobilized bacteria, yeast and other fungi, as well as activatedsludge; microcarrier bioreactors are commonly used with mammalian cellsimmobilized on solid particles; surface tissue propagators are commonlyused with mammalian cells, tissue grown on solid surfaces, and tissueengineering; membrane bioreactors, hollow fibers and roto-fermentors aretypically used with bacteria, yeast, mammalian cells, and plant cells;modified stir-tank bioreactors are commonly used with immobilizedbacteria, yeast, and plant cells; modified packed-bed bioreactors arecommonly used with immobilized bacteria, yeast, and other fungi; towerand loop bioreactors are commonly used with bacteria and yeast; vacuumand cyclone bioreactors are commonly used with bacteria, yeast, andfungi; and photochemical bioreactors are commonly used withphotosynthetic bacteria, algae, cyanobacteria, plant cell culture,and/or DNA plant cells.

Since living cells, including bacteria, yeast, plant cells, mammaliancells, and fungal cells require molecular oxygen as an electron acceptorin the bioxidation of substrates (such as sugars, fats, and proteins),cell culture media that is highly oxygenated is beneficial to the livingcells. In a standard oxidation-reduction reaction, glucose is oxidizedto make carbon dioxide, while oxygen is reduced to make water. Molecularoxygen accepts all of the electrons released from the substrates duringaerobic metabolism. Thus, in order to provide the maximum amount ofbio-available oxygen to the growing cells, it is necessary to ensurethat the oxygen transfer from the air bubbles (gas phase) to the liquidphase occurs quickly. When no oxygen accumulates in the liquid phase,the rate of the oxygen transfer is the same as the rate of the oxygenuptake by the growing cells.

The oxygen requirements of microorganisms is defined as a standardformula, that is in units of QO₂. Where QO₂ is the mass of oxygenconsumed divided by the unit weight of dry biomass cells in thebioreactor multiplied by time. Conversely, the rate of accumulation ofoxygen is equal to the net rate of oxygen supply from air bubbles minusthe rate of oxygen consumption by cells.

In addition to a multitude of bioreactor types, each bioreactor mayutilize a particular impeller type or types, such as marine-typepropellers, flat-blade turbines, disk flat-blade turbines, curved-bladeturbines, pitched-blade turbines, paddles, and shrouded turbines. Theimpeller or turbine may create a vortex pattern of mixing in thebioreactor, or a uniform mixing.

In certain embodiments, the gas-enriched fluid of the present inventionrelates to a sustained bio-availability of the gas such that a gradualrelease of the gas occurs over time. This gradual or “time” release isbeneficial to the vehicles, such as cultured cells, particularly whenthe gas released from the gas-enriched fluid comprises oxygen. Thus,fermentation, or the biochemical synthesis of organic compounds by cells3310, typically involve a relatively fast growth phase, facilitated bythe concentrations of diffused or dissolved gas in the broth 3312, aswell as by temperature control and by mixing the medium 3304 and thecell culture 3310 in the fermentation chamber of the bioreactor 3306.Particular exemplary embodiments are depicted in the figures, but mayinclude additional components or tanks. Mixing may be enhanced byrotating vanes or the like within bioreactor 3306, and by reintroductionof fresh and/or freshly re-diffused supplies of medium 3304 from any ofthe lines 3332, 3338 or 3334, as described herein inter alia.

In one particular exemplary embodiment depicted in FIG. 45A, theenrichment processing of the medium and/or broth to introduce the gas(e.g. oxygen) in a cell culture medium may occur at various points inthe system. For example, the medium may be gas-enriched prior tointroducing the medium 3304 into the system 3300 a at source 3302, orafter such introduction at one or more locations “A,” “B,” “C,” “D,” “E”or combinations thereof. Gas-enriched fluid that may be introduced atthe source 3302, whether enriched at the site of the bioreactor or at aseparate location. If the gas-enriched fluid is enriched at a separatelocation, it may be transported to the source 3302 in appropriatecontainers or plumbing.

In certain embodiments, each of the locations “A,” “B,” and “C,” of FIG.45A represent alternative locations for introduction of a gas-enrichmentdiffuser device 1-within the bioreactor system 3300A. In the event thatthe introduction occurs at point “A,” the flow of medium from tank 3304through the upper section of 3332A of 3332, the medium may be directedthrough the gas-enrichment mixer/diffuser device 100 located at position“A,” and medium 3304 with dissolved gases therein proceeds from themixer/diffuser device 100 through 3332B and into the fermentationchamber of the bioreactor 3306.

With reference to FIG. 45B, the medium 3304 from tank 3302 may bedirected through line 3332A into a pump 3410 and, subsequently into thehost material input of the gas-enrichment diffuser device 100. The pump3410, is preferably a variable speed pump, which may be controlled by acontroller 3390, based, in part, on pressure readings detected bypressure sensor 3415. While certain embodiments will utilize manualgauges as pressure detectors, from which an operator may manually adjustthe speed of the pump 3410, and other components of the system 3300 a or3300 b, controller 3390 preferably receives an electrical signal fromsensor 3415 such that controller 3390 will automatically adjust thespeed of pump 3410. As will be evident from further descriptions herein,the speed of pump 3410 may also be based on algorithms within thecontroller 3390 which depend, in part, on the state of other componentsof the system 3400 (such as valves 3420, 3421 and sensors 3425).

Alternatively, the gas-enrichment mixer/diffuser device 100 may bepositioned at location “B” such that the medium 3304 is processedtogether with medium 3310. In this particular case, cells 3310 andmedium 3304 are mixed in flow using a conventional mixing nozzle andsubsequently introduced into the mixer/diffuser device 100, wherebeneficial gases are infused into the mixed liquid of medium 3304 andcells 3310. The resulting gas-enriched medium is then directed into thefermentation tank of the bioreactor 3306.

As shown in FIG. 45A, cells 3310 may be combined with medium 3304, andfollowing fermentation and/or development of the target product, thecontents of the bioreactor 3306 may then be directed through line 3336to a harvesting and purification stage. Once purified, the targetproduct is directed through line 3339 to a target production tank 3318.

With reference to FIG. 45B, in certain embodiments, the gas-enrichmentmixer/diffuser device 100 combines the flow of a medium 3304 with a flowof a gas from line 3426. Preferably, the gas to be combined with medium3304 flows from an oxygen tank 3450 and is metered by a valve 3420,which is controlled by controller 3390.

In certain embodiments, the gas-enrichment mixer/diffuser device 100 isdirected through line 3332 b directly into the fermentation tank by areactor 3306. Alternatively, the gas-enriched fluid may be directedthrough line 3332 b to another blending.

With reference to FIG. 45A, a bioreactor system 3300 a, may include anadditional system (such as a perfusion system) 3314 that beginsprocessing the broth from the bioreactor 3306. During the perfusionprocess 3314, the medium 3304 is continuously added to the broth 3312 tonourish the cell culture 3310, which is then mixed throughout the broth3312. Simultaneously, cell or other waste may be continuously removedfrom the broth 3312, typically at the same rate as new medium 3304 isadded. As indicated herein above, gas-enrichment may also occur atpositions “D” or “E,” or at both positions “D” and “E.”

The perfusion system can allow for removal of cellular waste and debris,as well as the target product, while retaining the cells in thebioreactor 3306. The perfusion system thus reduces waste accumulationand nutrient fluctuations, thereby allowing for higher cell density andproductivity. Retention of the cells in the bioreactor may be achievedthrough various methods, including centrifugation, internal or externalspin filters, hollow fiber modules, cross-flow filtration, depthfiltration, any combination of these or other means. In otherembodiments, the accumulation of waste products may be regulated by useof a glutamine synthetase expression system.

With reference to FIG. 46, particular exemplary embodiments utilizemultiple gas sources 3502 and 3504 as shown, such that the nature of thegas being diffused into the broth 3312 can be changed depending on thestage of fermentation within the bioreactor 3306. Hence, in a preferredembodiment, the cell culture medium is enriched with oxygen during theproliferative phase of fermentation. Subsequently, carbon dioxide,nitrous oxide, or another gas may be substituted to facilitate otherstages of the fermentation process, particularly with processes thatvary from aerobic to anaerobic.

The bioreactor may comprise an airlift reactor, a packed bed reactor, afibrous bed reactor, a membrane reactor, a two-chamber reactor, astirred-tank reactor, a hollow-fiber reactor, or other reactor designedto support suspended or immobilized cell growth.

In one particular embodiment, the bioreactor 3306 is a continuousstirred-tank reactor, comprising heat exchange and refrigerationcapabilities, sensors, controllers, and/or a control system to monitorand control the environmental conditions within the fermentationchamber. Monitored and controlled conditions may include gas (e.g. air,oxygen, nitrogen, carbon dioxide, nitrous oxide, nitric oxide, sulfurgas, carbon monoxide, hydrogen, argon, helium, flow rates, temperature,pH, dissolved oxygen levels, agitation speed, circulation rate, andothers. Additionally, the bioreactor 3306 may further compriseCleaning-in-Place (CIP) or Sterilization-in-Place (SIP) systems, whichmay be cleaned and/or sterilized without assembly or disassembly of theunits.

In one particular embodiment, the bioreactor 3306 performs a continuousfermentation cycle, continuously adding medium 3304 to the fermentationsystem with a balancing withdrawal, or harvest, of the broth 3312 fortarget product extraction.

In alternate embodiments, the bioreactor 3306 may perform batchfermentation cycles, fed-batch fermentation cycles, or fed-batchfermentation cycles with the gas-enriched fluids. Typically, batchfermentation cycles—in which all of the reactants are loadedsimultaneously—are used for small scale operations or for themanufacture of expensive products or for processes that may be difficultto convert into continuous operations. In a typical process, the brothis fermented for a defined period to completion, without furtheradditions of the medium. The concentration varies with time, but istypically uniform at any one particular time point. Agitation serves tomix separate feed lines as well as enhance heat transfer.

For batch fermentation, typically the total mass of each batch is fixed,each batch is a closed system, and the reaction or residence time forall reactants of the medium is the same. After discharging the batch,the fermentation chamber is cleaned and re-started with the medium 3304for another batch cycle. Separation or purification of the desiredproduct from the other constituents in the harvest broth 3312, mayinclude further processing, including refolding, altering affinity, ionexchange purification, alteration of hydrophobic interactions, gelfiltration chromatography, ultra filtration and/or diafiltration,depending on the target product.

For fed-batch fermentation, typically an initial, partial charge oraliquot of medium 3304 is added to the fermentation chamber, andsubsequently inoculated with cell culture 3304. The medium 3304 may beadded at measured rates during the remainder of the fermentation cycle.The cell mass and the broth 3312 are typically harvested only at the endof the cycle.

Following harvest and purification of the target product (step 3316),(typically once the cell culture 3310 has attained a peak cell growthdensity within the bioreactor 3306), the purified product 3318 (in somecases, a pharmaceutical drug or Active Pharmaceutical Ingredient, orAPI) is attained. The purified product may then be processed as desiredand optionally packaged in appropriate containers during a sterilepackaging process 3322 for transfer to a pharmaceutical manufacturingplant, or other facility. The purified product may then be used for anydesired purpose, including for prevention, treatment, and/or diagnosisof disease.

Plants and Animals as Reactors

In addition, a reactor may include a plant or animal, which is used togenerate a plant or animal product, or recombinant product. In certainembodiments, the plant or animal target product may be a naturallyoccurring product (e.g., food bearing crops or meat, or textile-relatedproducts such as cotton fibers, etc.), or the target product may be agenetically altered product (for example, therapeutic agents, such humangrowth hormone or insulin or other biologically active proteins andpolypeptides). A genetically altered or recombinant product may beproduced by a transgenic or genetically altered plant, animal, orcombination thereof.

Fish Culture

Fish (e.g., Tilapia fish) may be grown in aquaculture for food, or as atransgenic vehicle for production of a target product. The preferredtemperature range for optimum tilapia growth is 82°-86° F. Growthdiminishes significantly at temperatures below 68° F. and death willtypically occur below 50° F. Also, at temperatures below about 54° F.,the immune resistance of tilapia declines and the animals are easilysubjected to infection by bacteria, fungi, and parasites.

Twenty years ago, aquaculture researchers in Nigeria attempted tocorrelate dissolved oxygen concentrations in pond waiter with Tilapiagrowth rates. UN FAO reports: The study was conducted by examininggrowth rates of young Tialapia at high dissolved oxygen levels(approximately 7.0 ppm); at mid-level DO (approximately 3.5 ppm); and atlow DO levels (less than 2 ppm). The growth rates were determined bymeasuring the weight of the fish. The final increase in weight at theend of the research was 19 grams for the high DO level fish; 5 grams forthe mid-level DO fish; and 1.5 g for the low DO level fish. Thisrepresents to a 74% and 92% reduction in growth rates correlating to theDO levels. Thus, as the DO levels decrease, the feeding and waste outputalso decrease. It was observed that the Tilapia in the low DO levelwater break the surface of the water in order to access ambient oxygenrequired for survival.

The gas-enriched fluids of the present invention further includeoxygenated freshwater supplies in which the high dissolved oxygen levelsin the water are maintained for extended periods of time. According toparticular aspects, using the diffuser device of the present inventionin an aquaculture setting, dissolved oxygen levels of over 35 ppm can berecorded in 103° F. water without significantly stressing the aquaticlife.

Plant Growth

In addition to animal growth, the gas-enriched fluids of the presentinvention may be utilized for plant growth and development. Gases (suchas oxygen) are required for plant root respiration, which allows for therelease of energy for growth, as well as water and mineral uptake. Plantgrowth has been widely and unequivocally proven to be boosted bymaintaining high gas (e.g. oxygen and/or nitrogen) levels within theroot zone. In this regard, increasing gas delivery to plant root systemsrepresents a potential for crop improvement through boosting rootactivity. Likewise, in embodiments in which transgenic plants are grown,increasing gas delivery to the plants may provide for increasedproduction of the target product (such as a therapeutic orbiopharmaceutical product).

Hydroponic crops represent one exemplary system for production which maygreatly benefit from the gas-enrichment diffuser devices of the presentinvention through direct gas-enrichment (e.g. oxygenation) of thenutrient solution bathing the root zone. Hydroponic crops are typicallyproduced in a limited volume of growing media or root area and as suchneed constant replacement of gases (e.g. oxygen) within the root zone.Hydroponic crops such as lettuce, spinach, tomatoes, and cucumbers havealready demonstrated a direct and significant response to thegas-enriched nutrient solution. Some of these responses includeincreases in plant growth, increases in root volume, increases in plantyield, and higher quality produce. Thus, hydroponic systems may benefitfrom the gas-enriched fluids of the present invention.

Other hydroponic crops have had similar responses to gas-enrichment inthe root zone. However, at warm temperatures, crop production declinesdue to the increased requirement for gases (such as oxygen) in the rootzone. Thus, enrichment is effective for preventing gas-starvation ofroot cells, as well as boosting growth under less than favorable growingconditions.

Typically tropical crops that are able to be grown at high densities dueto high light levels and rapid rates of development (and high root zonetemperatures) have a gas requirement that is many times greater thanthose grown in more temperate climates. Thus, gas-enrichment will becomenecessary in many systems of horticulture production. Highly populatedcountries, which rely heavily on producing intensive horticultural cropsfor income and sustenance from very limited areas of land, will benefitgreatly from this technology.

Soil-based cropping systems can also benefit from the gas-enrichedsolutions of the present invention. Many crops are fed via drip,trickle, or furrow irrigation and could potentially benefit greatly fromthe use of gas-enriched irrigation water or fertigation solutions. Suchcrops include, but are not limited to: vegetables (tomatoes, salad cropssuch as lettuce, herbs, cucurbits), cut flowers, ornamental flowers,turf, vineyards, orchards, and long-term plantings. Gases, such asoxygen, can directly impact the health and growth of the plant but canalso act indirectly by increasing the bio-availability of gases (e.g.oxygen) at the root zone, and can also improve the health of the plantby promoting microbial life in the soil.

With regard to the microbial life in the soil, the microbial populationsare essential for mineral conversion in the soil and organic systems andoverall plant health through suppression of plant diseases. While thesemicrobes are beneficial and often essential for crop production, thepopulations also require gases (e.g. oxygen), which can compete with thegases for plant root cells. Thus, supplying gases (e.g. oxygen) to theplant roots in order to enable microbial life to flourish is vital toboth organically grown crops, as well as standard growing conditions.High rates of gases supplied to the growing media/soil in organicsystems would potentially speed up the rate of organic fertilizerconversion and mineralization of plant usable nutrients, thus increasingthe health and productivity of highly profitable organic crops.

In addition, the available land for growing crops represents a challengein many countries with limited resources or unsuitable soils.

In addition to hydroponic crops, the technology disclosed herein mayapply to seed germination, seed raising, cell transplant production,propagation from cuttings, sprout production, animal fodder production,soil based cropping, turf industries, ornamental plants, and medicinalplants.

Systems for Making Gas-Enriched Fluids

As shown here, exemplary oxygenation systems comprises a supply orreservoir of fluid which is drawn up and circulated through tubing orother conduits by a pump which subsequently delivers the fluid to themixer/diffuser. The mixer/diffuser may be of any number of variousembodiments including those set forth and described herein above. Thesediffusers significantly increase the amount of dissolved gas (e.g.oxygen) present in a fluid by introducing, for example, gaseous oxygento the fluid using a diffuser having coaxial cylindrical or frustoconical stator and rotor components rotating discs or plates within ahousing, Mazzie diffusers and impellers to create the desired cavitationand succussion desired for mixing of the fluid and the gas. It should benoted that many of the fluids will be aqueous or water-based, but thatthe present invention is not limited to these.

The diffuser is supplied with fluid by the pump and combines this with,for example, gaseous oxygen from supply and returns the oxygenated (orotherwise gas-enriched) fluid to the reservoir. The diffuser may employany number of possible embodiments for achieving diffusion including,but not limited to, micro-membrane, Mazzie injector, fine bubble,vortexing, electromolecular activation, or other methods. The oxygensupply may be either a cylinder of compressed oxygen gas or a system forgenerating oxygen gas from the air or other chemical components. Theoxygenated fluid produced by the diffuser is returned to the reservoirand may be recirculated through the pump and/or the diffuser again tofurther increase the dissolved oxygen content. Alternatively, the fluidmay be drawn off using the oxygenated fluid outlet. Oxygenated fluidswhich are drawn off through the outlet may be immediately put to use invarious applications or may be packaged for later use.

The packaging step may enclose gas-enriched (e.g. oxygenated) fluids ina variety of bottles, bags or other containers formed of plastic, metal,glass, or other suitable materials. Although the gas-enriched oroxygenated fluids produced in accordance with the present invention havea relatively long shelf life under atmospheric conditions, the shelflife may be further extended by using packaging which hermetically sealsthe gas-enriched fluid. In this manner, dissolved oxygen which works itsway out of the fluid during storage will form a pressure head above thegas-enriched fluid and help to prevent the migration of dissolvedoxygen, or other gas, out of the fluid and back into the atmosphere. Inone preferred embodiment of the present invention the gas-enriched fluidis packaged in an air tight container and the void space is filled withthe gas used for enrichment at a pressure of greater than one atmosphereprior to sealing the container. The packaging step may be used toproduce bottles, bags, pouches, or other suitable containers for holdingoxygenated solutions.

The presently disclosed systems and/or methods allow oxygen, or othergases, to be dissolved stably at a high concentration with minimalpassive loss. These systems and/or methods can be effectively used todissolve a wide variety of gases at heightened percentages into a widevariety of fluids. By way of example only, a deionized water at roomtemperature that typically has levels of about 7-9 ppm (parts permillion) of dissolved oxygen can achieve levels of dissolved oxygenranging from about 8-70 ppm using the disclosed systems and/or methods.In accordance with a particular exemplary embodiment, an oxygenatedwater or saline solution may be generated with levels of about 30-60 ppmof dissolved oxygen.

Culturing Chinese Hamster Ovary Cells

Chinese Hamster Ovary (CHO) cells are mammalian cells that arefrequently utilized in expression and production of recombinantproteins, particularly for those that require post-translationalmodification to express full biological function.

According to particular aspects, various characteristics of CHO cellscan be improved by integrating either a gas-enriching diffuser device100 or gas-enriched media produced by the device 100 and integrated intoa CHO bioreactor.

According to particular aspects, in the cultivation of CHO cells, it ispossible to utilize the gas-enriched fluids or media of the presentinvention including with a cell-line specific, serum-free medium (forexample from SAFC Biosciences, Inc.) for long-term growth of transformedCHO cells. According to additional aspects, CHO cells are not harmed bypassing through the gas-enrichment diffuser device in the process ofgas-enriching fluids (including media).

A test was conducted that measured the survival of CHO cells in aninline bioreactor. Briefly, the inline bioreactor was used with 2 L ofCHO media, and CHO cells at a density of 10⁶ or higher. The bioreactorwas run for approximately 10 minutes (including the gas-enrichingdiffuser), and a 25 mL sample was removed. Cells were stained with 0.4%Trypan Blue, and cell viability was assessed with a hemacytometer.According to this measure, CHO cells were not significantly harmed bypassing through the gas-enrichment diffuser device in the process ofgas-enriching fluids (including media).

The foregoing described embodiments depict different componentscontained within, or connected with, different other components. It isto be understood that such depicted architectures are merely exemplary,and that in fact many other architectures can be implemented whichachieve the same functionality. In a conceptual sense, any arrangementof components to achieve the same functionality is effectively“associated” such that the desired functionality is achieved. Hence, anytwo components herein combined to achieve a particular functionality canbe seen as “associated with” each other such that the desiredfunctionality is achieved, irrespective of architectures or intermedialcomponents. Likewise, any two components so associated can also beviewed as being “operably connected,” or “operably coupled,” to eachother to achieve the desired functionality.

While particular embodiments of the present invention have been shownand described, it will be obvious to those skilled in the art that,based upon the teachings herein, changes and modifications may be madewithout departing from this invention and its broader aspects and,therefore, the appended claims are to encompass within their scope allsuch changes and modifications as are within the true spirit and scopeof this invention. Furthermore, it is to be understood that theinvention is solely defined by the appended claims. It will beunderstood by those within the art that, in general, terms used herein,and especially in the appended claims (e.g., bodies of the appendedclaims) are generally intended as “open” terms (e.g., the term“including” should be interpreted as “including but not limited to,” theterm “having” should be interpreted as “having at least,” the term“includes” should be interpreted as “includes but is not limited to,”etc.). It will be further understood by those within the art that if aspecific number of an introduced claim recitation is intended, such anintent will be explicitly recited in the claim, and in the absence ofsuch recitation no such intent is present. For example, as an aid tounderstanding, the following appended claims may contain usage of theintroductory phrases “at least one” and “one or more” to introduce claimrecitations. However, the use of such phrases should not be construed toimply that the introduction of a claim recitation by the indefinitearticles “a” or “an” limits any particular claim containing suchintroduced claim recitation to inventions containing only one suchrecitation, even when the same claim includes the introductory phrases“one or more” or “at least one” and indefinite articles such as “a” or“an” (e.g., “a” and/or “an” should typically be interpreted to mean “atleast one” or “one or more”). the same holds true for the use ofdefinite articles used to introduce claim recitations. In addition, evenif a specific number of an introduced claim recitation is explicitlyrecited, those skilled in the art will recognize that such recitationshould typically be interpreted to mean at least the recited number(e.g., the bare recitation of “two recitations,” without othermodifiers, typically means at least two recitations, or two or morerecitations).

Accordingly, the invention is not limited except as by the appendedclaims.

The invention claimed is:
 1. An electrokinetically altered composition,comprising an ionic aqueous solution of charge-stabilizedoxygen-containing nanostructures having an average diameter of less than100 nanometers and stably configured to persist for at least days in theionic aqueous solution in a sealed container at atmospheric pressure. 2.The composition of claim 1, wherein the composition comprises dissolvedoxygen present in an amount of at least 10 ppm oxygen at atmosphericpressure.
 3. The composition of claim 1, wherein the amount ofcharge-stabilized oxygen-containing nanostructures is at least 10 ppm atatmospheric pressure.
 4. The composition of claim 1, comprising at leastone of electrokinetically modified or charged oxygen species, andsolvated electrons stabilized by molecular oxygen.
 5. The composition ofclaim 4, wherein the solvated electrons are present in an amount of atleast 0.01 ppm.
 6. The composition of any one of claims 1-5, wherein thecharge-stabilized oxygen-containing nanostructures comprise at least oneof nanocages, nanoclusters and nanobubbles.
 7. The composition of anyone of claims 1-5, comprising saline or oxygen enriched saline, andoptionally wherein the charge-stabilized oxygen-containingnanostructures comprise at least one of nanocages, nanoclusters andnanobubbles.
 8. The composition of claim 1, wherein there is no hydrogenperoxide, or less than 0.1 ppm of hydrogen peroxide present in theelectrokinetically altered composition.
 9. The composition of claim 1,wherein the charge-stabilized oxygen-containing nanostructures persistsfor at least three hours in an open container, or for at least twomonths in a closed gas-tight container.
 10. A method of producing anelectrokinetically altered composition, comprising: providing a flow ofa fluid material between two spaced surfaces in relative motion anddefining a mixing volume therebetween, wherein the dwell time of asingle pass of the flowing fluid material within and through the mixingvolume is greater than 0.06 seconds; and introducing oxygen (O₂) intothe flowing fluid material within the mixing volume under conditionssuitable to dissolve at least 10 ppm oxygen into the material, toprovide an ionic aqueous solution of charge-stabilized oxygen-containingnanostructures having an average diameter of less than 100 nanometersand stably configured to persist for at least days in the ionic aqueoussolution in a sealed container at atmospheric pressure.
 11. The methodof claim 10, wherein the oxygen is infused into the material in lessthan 400 milliseconds.
 12. A method of producing an electrokineticallyaltered composition, comprising: providing a flow of a fluid materialbetween two spaced surfaces defining a mixing volume therebetween; andintroducing oxygen into the flowing material within the mixing volumeunder conditions suitable to infuse at least 10 ppm oxygen into thematerial in less than 400 milliseconds, to provide an ionic aqueoussolution of charge-stabilized oxygen-containing nanostructures having anaverage diameter of less than 100 nanometers and stably configured topersist for at least days in the ionic aqueous solution in a sealedcontainer at atmospheric pressure.
 13. The method of claim 12, whereinthe dwell time of the fluid material flowing within the mixing volume isgreater than 0.06 seconds.
 14. The method of any one of claims 10, 11,12 and 13, wherein the ratio of surface area to the volume is at least12.
 15. A method of producing an electrokinetically altered composition,comprising use of a mixing device for creating an output mixture bymixing a first material and a second material, the device comprising: afirst chamber configured to receive the first material from a source ofthe first material; a stator; a rotor having an axis of rotation, therotor being disposed inside the stator and configured to rotate aboutthe axis of rotation therein, at least one of the rotor and statorhaving a plurality of through-holes; a mixing chamber defined betweenthe rotor and the stator, the mixing chamber being in fluidcommunication with the first chamber and configured to receive the firstmaterial therefrom, and to receive the second material into the mixingchamber via the plurality of through-holes formed in the one of therotor and stator; a second chamber in fluid communication with themixing chamber and configured to receive the output material therefrom;and a first internal pump housed inside the first chamber, the firstinternal pump being configured to pump the first material from the firstchamber into the mixing chamber, wherein the first material comprises anaqueous material and the second material comprises oxygen to provide anionic aqueous solution of charge-stabilized oxygen-containingnanostructures having an average diameter of less than 100 nanometersand stably configured to persist for at least days in the ionic aqueoussolution in a sealed container at atmospheric pressure.
 16. A method ofproducing an electrokinetically altered composition, comprising use of amixing device for creating an output mixture by mixing a first materialand a second material, the device comprising: a stator; a rotor havingan axis of rotation, the rotor being disposed inside the stator andconfigured to rotate about the axis of rotation therein; a mixingchamber defined between the rotor and the stator, the mixing chamberhaving an open first end through which the first material enters themixing chamber and an open second end through which the output materialexits the mixing chamber, the second material entering the mixingchamber through at least one of the rotor and the stator; a firstchamber in communication with at least a majority portion of the openfirst end of the mixing chamber; and a second chamber in communicationwith the open second end of the mixing chamber, wherein the firstmaterial comprises an aqueous material and the second material comprisesoxygen to provide an ionic aqueous solution of charge-stabilizedoxygen-containing nanostructures having an average diameter of less than100 nanometers and stably configured to persist for at least days in theionic aqueous solution in a sealed container at atmospheric pressure.17. The method of claim 15, wherein the first internal pump isconfigured to impart a circumferential velocity into the first materialbefore it enters the mixing chamber.
 18. The composition of claim 1,wherein at least 90% of oxygen present in the electrokinetically-alteredaqueous fluid composition is in the charge-stabilized oxygen-containingnanostructures having an average diameter of less than 100 nanometers.19. The electrokinetically altered composition of any of claim 1-5,wherein the charge-stabilized oxygen-containing nanostructures arepresent in an amount sufficient to provide a beneficial advantage toliving cells contacted by the composition.
 20. The electrokineticallyaltered composition of claim 1, wherein the charge-stabilizedoxygen-containing nanostructures are present in an amount sufficient toprovide a beneficial advantage to living cells contacted by the fluid orsolution.
 21. The electrokinetically altered composition of any one ofclaim 1-5, wherein the composition comprises a beverage or beveragecomponent.
 22. The electrokinetically altered composition of any one ofclaim 1-5, wherein the composition comprises a beverage or beveragecomponent, and wherein the charge-stabilized oxygen-containingnanostructures comprise at least one of nanocages, nanoclusters andnanobubbles.
 23. The electrokinetically altered composition of any oneof claim 1-5, wherein the charge-stabilized oxygen-containingnanostructures comprise charge-stabilized oxygen-containing nanobubbleshaving an average diameter of less than 100 nanometers.